Nucleic acid molecules encoding an engineered antigen receptor and an inhibitory nucleic acid molecule and methods of use thereof

ABSTRACT

The present disclosure provides nucleic acid molecules encoding an engineered antigen receptor, such as a chimeric antigen receptor or exogenous T cell receptor, and an inhibitory nucleic acid molecule, such as an RNA interference molecule. The present disclosure further relates to nucleic acids, DNA constructs, vectors, pharmaceutical compositions, genetically-modified cells, and methods of treatment that utilize the nucleic acid molecules of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.16/927,452, filed Jul. 13, 2020, which is a Continuation of U.S.application Ser. No. 16/678,600, filed Nov. 8, 2019, which is aContinuation of PCT/US2018/031674 filed May 8, 2018, which InternationalApplication was published by the International Bureau in English on Nov.15, 2018, and application claims priority from U.S. Provisional PatentApplication No. 62/503,060, filed May 8, 2017, and U.S. ProvisionalPatent Application No. 62/579,460, filed Oct. 31, 2017, whichapplications are hereby incorporated in their entirety by reference inthis application.

FIELD OF THE INVENTION

The present disclosure relates to the field of molecular biology andrecombinant nucleic acid technology. In particular, the presentdisclosure relates to nucleic acid molecules encoding an engineeredantigen receptor, such as a chimeric antigen receptor or exogenous Tcell receptor, and an inhibitory nucleic acid molecule, such as an RNAinterference molecule. The present disclosure further relates to nucleicacids, DNA constructs, viral vectors, pharmaceutical compositions,genetically-modified cells, and methods of treatment that utilize thenucleic acid molecule of the invention.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 14, 2020, isnamed P89339_1050US_C1_Seq_List, and is 188513 bytes in size.

BACKGROUND OF THE INVENTION

T cell adoptive immunotherapy is a promising approach for cancertreatment. This strategy utilizes isolated human T cells that have beengenetically-modified to enhance their specificity for a specific tumorassociated antigen. Genetic modification may involve the expression of achimeric antigen receptor (CAR) or an exogenous T cell receptor to graftantigen specificity onto the T cell. By contrast to exogenous T cellreceptors, CARs derive their specificity from the variable domains of amonoclonal antibody. Thus, T cells expressing CARs induce tumorimmunoreactivity in a major histocompatibility complex (MHC)non-restricted manner. To date, T cell adoptive immunotherapy has beenutilized as a clinical therapy for a number of cancers, including B cellmalignancies (e.g., acute lymphoblastic leukemia (ALL), B cellnon-Hodgkin lymphoma (NHL), and chronic lymphocytic leukemia), multiplemyeloma, neuroblastoma, glioblastoma, advanced gliomas, ovarian cancer,mesothelioma, melanoma, and pancreatic cancer.

Despite its potential usefulness as a cancer treatment, adoptiveimmunotherapy has been limited, in part, by alloreactivity between hosttissues and allogeneic CAR T cells. One cause of alloreactivity arisesfrom the presence of non-host MHC class I molecules on the cell surfaceof CAR T cells. MHC class I molecules consist of two polypeptide chains,a and β. In humans, the α chain consists of three subunits, α1, α2, andα3, which are encoded by polymorphic human leukocyte antigen (HLA) geneson chromosome 6. The variability of HLA loci, and the encoded α chainsubunits, can cause allogeneic CAR T cells to be seen by the host immunesystem as foreign cells because they bear foreign MHC class I molecules.As a result, CAR T cells administered to a patient can be subject tohost versus graft (HvG) rejection, where they are recognized and killedby the host's cytotoxic T cells.

The β chain of MHC class I molecules consists of beta-2 microglobulin,which is encoded by the non-polymorphic beta-2 microglobulin (B2M) geneon chromosome 15 (SEQ ID NO: 1). Beta-2 microglobulin is non-covalentlylinked to the a3 subunit and is common to all MHC class I molecules.Furthermore, expression of MHC class I molecules at the cell surfacerequires its association with beta-2 microglobulin. As such, beta-2microglobulin represents a logical target for suppressing the expressionof MHC class I molecules on CAR T cells, which could render the cellsinvisible to host cytotoxic T cells and reduce alloreactivity. However,complete knockout of beta-2 microglobulin expression may result in NKcell killing of CAR T cells due to the lack of cell surface MHC class Imolecules, which could prompt NK cells to recognize them as non-self andinitiate cytotoxic action.

Another cause of alloreactivity to CAR T cells is the expression of theendogenous T cell receptor on the cell surface. T cell receptorstypically consist of variable α and β chains or, in smaller numbers,variable γ and δ chains. The T cell receptor complexes with accessoryproteins, including CD3, and functions with cell surface co-receptors(e.g., CD4 and CD8) to recognize antigens bound to MEW molecules onantigen presenting cells. In the case of allogeneic CAR T cells,expression of endogenous T cell receptors may cause the cell torecognize host MHC antigens following administration to a patient, whichcan lead to the development of graft-versus-host-disease (GVHD).

To forestall alloreactivity, clinical trials have largely focused on theuse of autologous CAR T cells, wherein a donor's T cells are isolated,genetically-modified to incorporate a chimeric antigen receptor, andthen re-infused into the same subject. An autologous approach providesimmune tolerance to the administered CAR T cells; however, this approachis constrained by both the time and expense necessary to producepatient-specific CART cells after a patient's cancer has been diagnosed.

Thus, a need exists in the art for the development of allogeneic CAR Tcells which exhibit reduced allogenicity but, at the same time, avoid NKcell killing in vivo.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a nucleic acid moleculecomprising: (a) a first expression cassette comprising a nucleic acidsequence encoding an engineered antigen receptor; (b) a secondexpression cassette comprising a nucleic acid sequence encoding aninhibitory nucleic acid molecule; (c) a 5′ homology arm; and (d) a 3′homology arm; wherein the 5′ homology arm and the 3′ homology arm havehomology to chromosomal regions flanking a nuclease recognition sequencein a gene of interest.

In some embodiments, the inhibitory nucleic acid molecule is an RNAinterference molecule. In certain embodiments, the RNA interferencemolecule is a short hairpin RNA (shRNA), a small interfering RNA(siRNA), a hairpin siRNA, a microRNA (miRNA), a precursor miRNA, or anmiRNA-adapted shRNA. In particular embodiments, the RNA interferencemolecule is an shRNA.

In some embodiments the engineered antigen receptor is a chimericantigen receptor. In other embodiments, the engineered antigen receptoris an exogenous T cell receptor.

In some embodiments, the nuclease recognition sequence is an engineeredmeganuclease recognition sequence, a TALEN recognition sequence, a zincfinger nuclease (ZFN) recognition sequence, a CRISPR/Cas recognitionsequence, a compact TALEN recognition sequence, or a megaTAL recognitionsequence. In certain embodiments, the nuclease recognition sequence isan engineered meganuclease recognition sequence.

In some embodiments, the gene of interest is any gene of interest. Incertain embodiments, the gene of interest is a human T cell receptoralpha constant region gene. In particular embodiments the nucleaserecognition sequence is an engineered meganuclease recognition sequence.In certain embodiments, the engineered meganuclease recognition sequencecomprises SEQ ID NO: 1 in a human T cell receptor alpha constant regiongene.

In some embodiments, the first expression cassette further comprises apromoter which drives expression of the engineered antigen receptor. Incertain embodiments, the promoter is a JeT promoter.

In some embodiments, the second expression cassette further comprises apromoter which drives expression of the inhibitory nucleic acidmolecule. In certain embodiments, the promoter is a U6 promoter.

In some embodiments, the first expression cassette comprises apolyadenylation signal to terminate translation of the engineeredantigen receptor. In some embodiments, the second expression cassettecomprises a central polypurine tract and central terminator sequence(cPPT/CTS) sequence to terminate translation of the inhibitory nucleicacid.

In some embodiments, the first expression cassette and the secondexpression cassette are in the same orientation in the nucleic acidmolecule. In certain embodiments, the first expression cassette and thesecond expression cassette are in a 5′ to 3′ orientation relative to the5′ and 3′ homology arms. In some such embodiments, the first expressioncassette is 5′ upstream of the second expression cassette. In other suchembodiments, the second expression cassette is 5′ upstream of the firstexpression cassette.

In some embodiments, wherein the first expression cassette and thesecond expression cassette are in the same orientation in the nucleicacid molecule, the first expression cassette and the second expressioncassette are in a 3′ to 5′ orientation relative to the 5′ and 3′homology arms. In some such embodiments, the first expression cassetteis 5′ upstream of the second expression cassette. In other suchembodiments, the second expression cassette is 5′ upstream of the firstexpression cassette.

In some embodiments, the first expression cassette and the secondexpression cassette are in opposite orientations in the nucleic acidmolecule. In some such embodiments, the first expression cassette is ina 3′ to 5′ orientation and the second expression cassette is in a 5′ to3′ orientation relative to the 5′ and 3′ homology arms. In certainembodiments, the first expression cassette is 5′ upstream of the secondexpression cassette. In other embodiments, the second expressioncassette is 5′ upstream of the first expression cassette.

In particular embodiments, wherein the first expression cassette and thesecond expression cassette are in opposite orientations in the nucleicacid molecule, the first expression cassette is in a 5′ to 3′orientation and the second expression cassette is in a 3′ to 5′orientation relative to the 5′ and 3′ homology arms. In some suchembodiments, the first expression cassette is 5′ upstream of the secondexpression cassette. In other such embodiments, the second expressioncassette is 5′ upstream of the first expression cassette.

In some embodiments, the nucleic acid molecule comprises multiple copiesof the second expression cassette. In some such embodiments, the copiesare identical. In further embodiments, the copies include a promoter, acoding sequence for the inhibitory nucleic acid molecule, and asequence, such as a (cPPT/CTS) sequence, to terminate translation of theinhibitory nucleic acid molecule. In some such embodiments, the copiesof the second expression cassette are in tandem in the nucleic acidmolecule, and can be in the same orientation, or in oppositeorientations. In other such embodiments, the copies may not be intandem, and can be in the same orientation, or in opposite orientations.

In some embodiments, the nucleic acid molecule further comprises a 5′inverted terminal repeat and a 3′ inverted terminal repeat flanking thefirst expression cassette and the second expression cassette.

In some embodiments, the inhibitory nucleic acid molecule is inhibitoryagainst human beta-2 microglobulin.

In some embodiments, the inhibitory nucleic acid molecule is inhibitoryagainst a component of the MHC class I molecule. In certain embodiments,the inhibitory molecule is inhibitory against an MHC class I alpha-1(α1) domain, alpha-2 (α2) domain, alpha-3 (α3) domain, or against beta-2microglobulin.

In some embodiments, the inhibitory nucleic acid molecule is inhibitoryagainst human CD52.

In certain embodiments, the inhibitory nucleic acid molecule is an shRNAinhibitory against beta-2 microglobulin, wherein the shRNA has asequence comprising any one of SEQ ID NOs: 2-4. In particularembodiments, the shRNA has a sequence comprising SEQ ID NO: 2. In somesuch embodiments, the first expression cassette and the secondexpression cassette are in a 3′ to 5′ orientation relative to the 5′ and3′ homology arms, and wherein the first expression cassette is 5′upstream of the second expression cassette. In some such embodiments,the first expression cassette comprises: (i) a nucleic acid sequenceencoding a chimeric antigen receptor or an exogenous T cell receptor;(ii) a JeT promoter which drives expression of the chimeric antigenreceptor or the exogenous T cell receptor; and (iii) a polyA sequence;and the second expression cassette comprises: (iv) a nucleic acidsequence encoding the shRNA; (v) a U6 promoter which drives expressionof the shRNA; and (vi) a central polypurine tract and central terminatorsequence (cPPT/CTS) sequence.

In certain embodiments, the inhibitory nucleic acid molecule is an shRNAinhibitory against beta-2 microglobulin, wherein the shRNA has asequence comprising any one of SEQ ID NOs: 2-4. In particularembodiments, the shRNA has a sequence comprising SEQ ID NO: 2. In somesuch embodiments, the first expression cassette is in a 3′ to 5′orientation and the second expression cassette is in a 5′ to 3′orientation relative to the 5′ and 3′ homology arms, and the firstexpression cassette is 5′ upstream of the second expression cassette. Insome such embodiments, the first expression cassette comprises: (i) anucleic acid sequence encoding a chimeric antigen receptor or anexogenous T cell receptor; (ii) a JeT promoter which drives expressionof the chimeric antigen receptor or the exogenous T cell receptor; and(iii) a polyA sequence; and the second expression cassette comprises:(iv) a nucleic acid sequence encoding the shRNA; (v) a U6 promoter whichdrives expression of the shRNA; and (vi) a central polypurine tract andcentral terminator sequence (cPPT/CTS) sequence. In some suchembodiments, the nucleic acid molecule comprises a first copy and asecond copy of the second expression cassette, wherein the first copyand the second copy are identical, and wherein the first copy and thesecond copy are in tandem, and further wherein the first copy and thesecond copy are in the same orientation.

In another aspect, the invention provides a recombinant DNA constructcomprising any nucleic acid molecule of the invention described herein.

In some embodiments, the recombinant DNA construct encodes a viralvector. In certain embodiments, the viral vector is an adenoviralvector, a lentiviral vector, a retroviral vector, or an adeno-associatedviral (AAV) vector. In particular embodiments, the viral vector is arecombinant AAV vector.

In another aspect, the invention provides a viral vector comprising anynucleic acid molecule of the invention described herein. In certainembodiments, the viral vector is an adenoviral vector, a lentiviralvector, a retroviral vector, or an adeno-associated viral (AAV) vector.In particular embodiments, the viral vector is a recombinant AAV vector.

In another aspect, the invention provides a method for producing agenetically-modified eukaryotic cell, the method comprising introducinginto a cell any nucleic acid molecule of the invention described hereinand: (a) a nucleic acid encoding an engineered nuclease havingspecificity for the nuclease recognition sequence, wherein theengineered nuclease is expressed in the cell; or (b) an engineerednuclease protein having specificity for the nuclease recognitionsequence; wherein the engineered nuclease recognizes and cleaves thenuclease recognition sequence in the genome of the cell to generate acleavage site, and wherein the nucleic acid molecule of the invention isinserted into the genome of the cell at the cleavage site.

In some embodiments of the method, the genetically-modified eukaryoticcell is a human T cell.

In some embodiments of the method, the engineered nuclease is anengineered meganuclease, a TALEN, a zinc finger nuclease (ZFN), aCRISPR/Cas, a compact TALEN, or a megaTAL. In certain embodiments of themethod, the engineered nuclease is an engineered meganuclease.

In some embodiments of the method, the nuclease recognition sequence isin a human T cell receptor alpha constant region gene.

In certain embodiments of the method, the nuclease recognition sequenceis an engineered meganuclease recognition sequence. In particularembodiments, wherein the engineered meganuclease recognition sequence iswithin a human T cell receptor alpha constant region, the nucleaserecognition sequence comprises SEQ ID NO: 1.

In some embodiments of the method, wherein the nuclease recognitionsequence is within a human T cell receptor alpha constant region, cellsurface expression of an endogenous T cell receptor is reduced comparedto a control cell.

In some embodiments of the method, the nucleic acid encoding theengineered nuclease is an mRNA. In certain embodiments, the mRNA is apolycistronic mRNA encoding the engineered nuclease and at least oneadditional polypeptide or nucleic acid molecule.

In some embodiments of the method, the nucleic acid molecule of theinvention described herein is introduced into the cell using a viralvector. In certain embodiments of the method, the viral vector is anadenoviral vector, a lentiviral vector, a retroviral vector, or an AAVvector. In particular embodiments of the method, the viral vector is arecombinant AAV vector, such as a recombinant AAV vector previouslydescribed herein.

In some embodiments of the method, the nucleic acid molecule of theinvention described herein is introduced into the cell using arecombinant DNA construct. In certain embodiments of the method, therecombinant DNA construct is a recombinant DNA construct previouslydescribed herein.

In some embodiments of the method, the nucleic acid molecule of theinvention described herein is inserted into the genome of the cell atthe cleavage site by homologous recombination.

In some embodiments of the method, the engineered antigen receptor is achimeric antigen receptor. In other embodiments of the method, theengineered antigen receptor is an exogenous T cell receptor.

In some embodiments of the method, the inhibitory nucleic acid moleculeis inhibitory against human beta-2 microglobulin. In certain embodimentsof the method, cell surface expression of beta-2 microglobulin isbetween about 1% and about 50% of cell surface beta-2 microglobulinexpression on a control cell. In other embodiments of the method, cellsurface expression of beta-2 microglobulin is between about 1% and about25% of cell surface beta-2 microglobulin expression on a control cell.In other embodiments of the method, cell surface expression of beta-2microglobulin is between about 1% and about 10% of cell surface beta-2microglobulin expression on a control cell. In other embodiments of themethod, cell surface expression of beta-2 microglobulin is between about1% and about 5% of cell surface beta-2 microglobulin expression on acontrol cell. In particular embodiments of the method, a control cell isnot genetically-modified to reduce cell surface beta-2 microglobulinexpression.

In some embodiments of the method, the inhibitory nucleic acid moleculeis inhibitory against human beta-2 microglobulin. In certain embodimentsof the method, cell surface expression of beta-2 microglobulin isreduced by 10% to 95% compared to cell surface beta-2 microglobulinexpression on a control cell. In other embodiments of the method, cellsurface expression of beta-2 microglobulin is reduced by 50% to 95%compared to cell surface beta-2 microglobulin expression on a controlcell. In other embodiments of the method, cell surface expression ofbeta-2 microglobulin is reduced by 75% to 95% compared to cell surfacebeta-2 microglobulin expression on a control cell. In other embodimentsof the method, cell surface expression of beta-2 microglobulin isreduced by 90% to 95% compared to cell surface beta-2 microglobulinexpression on a control cell. In particular embodiments of the method, acontrol cell is not genetically-modified to reduce cell surface beta-2microglobulin expression.

In some embodiments of the method, the inhibitory nucleic acid moleculeis inhibitory against a component of the MHC class I molecule. Incertain embodiments of the method, cell surface expression of MHC classI molecules is between about 1% and about 50% of expression of MHC classI molecules on a control cell. In certain embodiments of the method,cell surface expression of MHC class I molecules is between about 1% andabout 25% of expression of MHC class I molecules on a control cell. Incertain embodiments of the method, cell surface expression of MHC classI molecules is between about 1% and about 10% of expression of MHC classI molecules on a control cell. In certain embodiments of the method,cell surface expression of MHC class I molecules is between about 1% andabout 5% of expression of MHC class I molecules on a control cell. Inparticular embodiments of the method, a control cell is notgenetically-modified to reduce cell surface expression of MHC class Imolecules.

In some embodiments of the method, the inhibitory nucleic acid moleculeis inhibitory against a component of the MHC class I molecule. Incertain embodiments of the method, cell surface expression of MHC classI molecules is reduced by 10% to 95% compared to expression of MHC classI molecules on a control cell. In certain embodiments of the method,cell surface expression of MHC class I molecules is reduced by 50% to95% compared to expression of MHC class I molecules on a control cell.In certain embodiments of the method, cell surface expression of MHCclass I molecules is reduced by 75% to 95% compared to expression of MHCclass I molecules on a control cell. In certain embodiments of themethod, cell surface expression of MHC class I molecules is reduced by90% to 95% compared to expression of MHC class I molecules on a controlcell. In particular embodiments of the method, a control cell is notgenetically-modified to reduce cell surface expression of MHC class Imolecules.

In some embodiments of the method, the inhibitory nucleic acid moleculeis inhibitory against human CD52. In certain embodiments of the method,cell surface expression of CD52 is between about 1% and about 50% ofcell surface CD52 expression on a control cell. In other embodiments ofthe method, cell surface expression of CD52 is between about 1% andabout 25% of cell surface CD52 expression on a control cell. In otherembodiments of the method, cell surface expression of CD52 is betweenabout 1% and about 10% of cell surface CD52 expression on a controlcell. In other embodiments of the method, cell surface expression ofCD52 is between about 1% and about 5% of cell surface CD52 expression ona control cell. In particular embodiments of the method, a control cellis not genetically-modified to reduce cell surface expression of CD52.

In some embodiments of the method, the inhibitory nucleic acid moleculeis inhibitory against human CD52. In certain embodiments of the method,cell surface expression of CD52 is reduced by 10% to 95% compared tocell surface CD52 expression on a control cell. In other embodiments ofthe method, cell surface expression of CD52 is reduced by 50% to 95%compared to cell surface CD52 expression on a control cell. In otherembodiments of the method, cell surface expression of CD52 is reduced by75% to 95% compared to cell surface CD52 expression on a control cell.In other embodiments of the method, cell surface expression of CD52 isreduced by 90% to 95% compared to cell surface CD52 expression on acontrol cell. In particular embodiments of the method, a control cell isnot genetically-modified to reduce cell surface expression of CD52.

In another aspect the invention provides a genetically-modifiedeukaryotic cell made by any of the methods described herein above.

In another aspect, the invention provides a genetically-modifiedeukaryotic cell comprising any nucleic acid molecule of the inventiondescribed herein, wherein the engineered antigen receptor and theinhibitory nucleic acid molecule are expressed in thegenetically-modified eukaryotic cell.

In some embodiments, the genetically-modified eukaryotic cell is agenetically-modified human T cell.

In some embodiments, the nucleic acid molecule of the invention isinserted into the genome of the genetically-modified eukaryotic cell atthe nuclease recognition sequence.

In some embodiments, the gene of interest is a human T cell receptoralpha constant region gene.

In some embodiments, the nuclease recognition sequence is an engineeredmeganuclease recognition sequence, a TALEN recognition sequence, a zincfinger nuclease (ZFN) recognition sequence, a CRISPR/Cas recognitionsequence, a compact TALEN recognition sequence, or a megaTAL recognitionsequence. In certain embodiments, the nuclease recognition sequence isan engineered meganuclease recognition sequence.

In particular embodiments, wherein the nuclease recognition sequence iswithin a human T cell receptor alpha constant region gene, the nucleaserecognition sequence is an engineered meganuclease recognition sequencecomprising SEQ ID NO: 1.

In some embodiments, cell surface expression of an endogenous T cellreceptor is reduced compared to a control cell.

In some embodiments, the inhibitory nucleic acid molecule is inhibitoryagainst human beta-2 microglobulin. In certain embodiments, cell surfaceexpression of beta-2 microglobulin is between about 1% and about 50% ofcell surface beta-2 microglobulin expression on a control cell. In otherembodiments, cell surface expression of beta-2 microglobulin is betweenabout 1% and about 25% of cell surface beta-2 microglobulin expressionon a control cell. In other embodiments, cell surface expression ofbeta-2 microglobulin is between about 1% and about 10% of cell surfacebeta-2 microglobulin expression on a control cell. In other embodiments,cell surface expression of beta-2 microglobulin is between about 1% andabout 5% of cell surface beta-2 microglobulin expression on a controlcell. In particular embodiments, a control cell is notgenetically-modified to reduce cell surface beta-2 microglobulinexpression.

In some embodiments, the inhibitory nucleic acid molecule is inhibitoryagainst human beta-2 microglobulin. In certain embodiments, cell surfaceexpression of beta-2 microglobulin is reduced by 10% to 95% compared tocell surface beta-2 microglobulin expression on a control cell. In otherembodiments, cell surface expression of beta-2 microglobulin is reducedby 50% to 95% compared to cell surface beta-2 microglobulin expressionon a control cell. In other embodiments, cell surface expression ofbeta-2 microglobulin is reduced by 75% to 95% compared to cell surfacebeta-2 microglobulin expression on a control cell. In other embodiments,cell surface expression of beta-2 microglobulin is reduced by 90% to 95%compared to cell surface beta-2 microglobulin expression on a controlcell. In particular embodiments, a control cell is notgenetically-modified to reduce cell surface beta-2 microglobulinexpression.

In some embodiments, the inhibitory nucleic acid molecule is inhibitoryagainst a component of the MHC class I molecule. In certain embodiments,cell surface expression of MHC class I molecules is between about 1% andabout 50% of expression of MHC class I molecules on a control cell. Incertain embodiments, cell surface expression of MHC class I molecules isbetween about 1% and about 25% of expression of MHC class I molecules ona control cell. In certain embodiments, cell surface expression of MHCclass I molecules is between about 1% and about 10% of expression of MHCclass I molecules on a control cell. In certain embodiments, cellsurface expression of MHC class I molecules is between about 1% andabout 5% of expression of MHC class I molecules on a control cell. Inparticular embodiments, a control cell is not genetically-modified toreduce cell surface expression of MHC class I molecules.

In some embodiments, the inhibitory nucleic acid molecule is inhibitoryagainst a component of the MHC class I molecule. In certain embodiments,cell surface expression of MHC class I molecules is reduced by 10% to95% compared to expression of MHC class I molecules on a control cell.In certain embodiments, cell surface expression of MHC class I moleculesis reduced by 50% to 95% compared to expression of MHC class I moleculeson a control cell. In certain embodiments, cell surface expression ofMHC class I molecules is reduced by 75% to 95% compared to expression ofMHC class I molecules on a control cell. In certain embodiments, cellsurface expression of WIC class I molecules is reduced by 90% to 95%compared to expression of MEW class I molecules on a control cell. Inparticular embodiments, a control cell is not genetically-modified toreduce cell surface expression of MEW class I molecules.

In some embodiments, the inhibitory nucleic acid molecule is inhibitoryagainst human CD52. In certain embodiments, cell surface expression ofCD52 is between about 1% and about 50% of cell surface CD52 expressionon a control cell. In other embodiments, cell surface expression of CD52is between about 1% and about 25% of cell surface CD52 expression on acontrol cell. In other embodiments, cell surface expression of CD52 isbetween about 1% and about 10% of cell surface CD52 expression on acontrol cell. In other embodiments, cell surface expression of CD52 isbetween about 1% and about 5% of cell surface CD52 expression on acontrol cell. In particular embodiments, a control cell is notgenetically-modified to reduce cell surface expression of CD52.

In some embodiments, the inhibitory nucleic acid molecule is inhibitoryagainst human CD52. In certain embodiments, cell surface expression ofCD52 is reduced by 10% to 95% compared to cell surface CD52 expressionon a control cell. In other embodiments, cell surface expression of CD52is reduced by 50% to 95% compared to cell surface CD52 expression on acontrol cell. In other embodiments, cell surface expression of CD52 isreduced by 75% to 95% compared to cell surface CD52 expression on acontrol cell. In other embodiments, cell surface expression of CD52 isreduced by 90% to 95% compared to cell surface CD52 expression on acontrol cell. In particular embodiments, a control cell is notgenetically-modified to reduce cell surface expression of CD52.

In another aspect, the invention provides a genetically-modifiedeukaryotic cell comprising in its genome a nucleic acid sequenceencoding an engineered antigen receptor which is expressed by thegenetically-modified eukaryotic cell, wherein cell surface expression ofbeta-2 microglobulin on the genetically-modified eukaryotic cell isreduced by 10% to 95% compared to cell surface beta-2 microglobulinexpression on a control cell. In certain embodiments, cell surfaceexpression of beta-2 microglobulin on the genetically-modifiedeukaryotic cell is reduced between 50% and 95% compared to cell surfacebeta-2 microglobulin expression on a control cell. In certainembodiments, cell surface expression of beta-2 microglobulin on thegenetically-modified eukaryotic cell is reduced between 75% and 95%compared to cell surface beta-2 microglobulin expression on a controlcell. In certain embodiments, cell surface expression of beta-2microglobulin on the genetically-modified eukaryotic cell is reducedbetween 90% and 95% compared to cell surface beta-2 microglobulinexpression on a control cell. In particular embodiments, the controlcell is not genetically-modified to reduce cell surface expression ofbeta-2 microglobulin.

In some embodiments, cell surface expression of beta-2 microglobulin isbetween about 1% and about 50% of cell surface beta-2 microglobulinexpression on a control cell. In other embodiments, cell surfaceexpression of beta-2 microglobulin is between about 1% and about 25% ofcell surface beta-2 microglobulin expression on a control cell. In otherembodiments, cell surface expression of beta-2 microglobulin is betweenabout 1% and about 10% of cell surface beta-2 microglobulin expressionon a control cell. In other embodiments, cell surface expression ofbeta-2 microglobulin is between about 1% and about 5% of cell surfacebeta-2 microglobulin expression on a control cell. In particularembodiments, a control cell is not genetically-modified to reduce cellsurface beta-2 microglobulin expression.

In certain embodiments, the genetically-modified eukaryotic cellcomprises in its genome a nucleic acid sequence encoding an inhibitorynucleic acid molecule which is inhibitory against beta-2 microglobulin.In particular embodiments, the inhibitory nucleic acid molecule is anRNA interference molecule. In some embodiments, the RNA interferencemolecule is a short hairpin RNA (shRNA), a small interfering RNA(siRNA), a hairpin siRNA, a microRNA (miRNA), a precursor miRNA, or anmiRNA-adapted shRNA. In certain embodiments, the RNA interferencemolecule is an shRNA. In particular embodiments, the shRNA comprises asequence of any one of SEQ ID NOs: 2-4. In specific embodiments, theshRNA comprises a sequence of SEQ ID NO: 2.

In some embodiments, the nucleic acid sequence encoding the engineeredantigen receptor is integrated at the same location within the genome asthe nucleic acid sequence encoding the inhibitory nucleic acid molecule.In particular embodiments, the genetically-modified eukaryotic cellcomprises in its genome the nucleic acid molecule of the invention.

In other embodiments, the nucleic acid sequence encoding the engineeredantigen receptor is integrated at a different location within the genomethan the nucleic acid sequence encoding the inhibitory nucleic acidmolecule.

In some embodiments, the genetically-modified eukaryotic cell is lesssusceptible to endogenous NK cell killing when compared to a controlcell, has extended persistence in a subject when compared to a controlcell, exhibits enhanced expansion in a subject when compared to acontrol cell, and/or exhibits reduced allogenicity when compared to acontrol cell.

In some embodiments, the engineered antigen receptor is a chimericantigen receptor or an exogenous T cell receptor.

In some embodiments, the genetically-modified eukaryotic cell is agenetically-modified human T cell.

In particular embodiments, the genetically-modified eukaryotic cell is agenetically-modified human T cell, and the engineered antigen receptoris a chimeric antigen receptor or an exogenous T cell receptor.

In another aspect, the invention provides a genetically-modifiedeukaryotic cell comprising in its genome a nucleic acid sequenceencoding an engineered antigen receptor which is expressed by thegenetically-modified eukaryotic cell, wherein cell surface expression ofMHC class I molecules on the genetically-modified eukaryotic cell isreduced by 10% to 95% compared to cell surface expression of MHC class Imolecules on a control cell. In certain embodiments, cell surfaceexpression of MHC class I molecules on the genetically-modifiedeukaryotic cell is reduced by 50% to 95% compared to cell surfaceexpression of MHC class I molecules on a control cell. In certainembodiments, cell surface expression of MHC class I molecules on thegenetically-modified eukaryotic cell is reduced by 75% to 95% comparedto cell surface expression of MHC class I molecules on a control cell.In certain embodiments, cell surface expression of MHC class I moleculeson the genetically-modified eukaryotic cell is reduced by 90% to 95%compared to cell surface expression of MHC class I molecules on acontrol cell. In particular embodiments, the control cell is notgenetically-modified to reduce cell surface expression of a component ofthe MHC class I molecule.

In some embodiments, cell surface expression of MHC class I molecules isbetween about 1% and about 50% of cell surface MHC class I moleculeexpression on a control cell. In other embodiments, cell surfaceexpression of MHC class I molecules is between about 1% and about 25% ofcell surface MHC class I molecule expression on a control cell. In otherembodiments, cell surface expression of MHC class I molecules is betweenabout 1% and about 10% of cell surface MHC class I molecule expressionon a control cell. In other embodiments, cell surface expression of MHCclass I molecules is between about 1% and about 5% of cell surface MHCclass I molecule expression on a control cell. In particularembodiments, a control cell is not genetically-modified to reduce cellsurface expression of MHC class I molecules.

In certain embodiments, the genetically-modified eukaryotic cellcomprises in its genome a nucleic acid sequence encoding an inhibitorynucleic acid molecule which is inhibitory against a component of the MHCclass I molecule. In certain embodiments, the inhibitory molecule isinhibitory against an MHC class I alpha-1 (□1) domain, alpha-2 (□2)domain, alpha-3 (□3) domain, or against beta-2 microglobulin. In aparticular embodiment, the inhibitory molecule is inhibitory againstbeta-2 microglobulin.

In particular embodiments, the inhibitory nucleic acid molecule is anRNA interference molecule. In some embodiments, the RNA interferencemolecule is a short hairpin RNA (shRNA), a small interfering RNA(siRNA), a hairpin siRNA, a microRNA (miRNA), a precursor miRNA, or anmiRNA-adapted shRNA. In certain embodiments, the RNA interferencemolecule is an shRNA.

In certain embodiments, the inhibitory nucleic acid molecule is an shRNAinhibitory against beta-2 microglobulin, wherein the shRNA has asequence comprising any one of SEQ ID NOs: 2-4. In particularembodiments, the shRNA has a sequence comprising SEQ ID NO: 2. In somesuch embodiments, the first expression cassette and the secondexpression cassette are in a 3′ to 5′ orientation relative to the 5′ and3′ homology arms, and wherein the first expression cassette is 5′upstream of the second expression cassette. In some such embodiments,the first expression cassette comprises: (i) a nucleic acid sequenceencoding a chimeric antigen receptor or an exogenous T cell receptor;(ii) a JeT promoter which drives expression of the chimeric antigenreceptor or the exogenous T cell receptor; and (iii) a polyA sequence;and the second expression cassette comprises: (iv) a nucleic acidsequence encoding the shRNA; (v) a U6 promoter which drives expressionof the shRNA; and (vi) a central polypurine tract and central terminatorsequence (cPPT/CTS) sequence.

In certain embodiments, the inhibitory nucleic acid molecule is an shRNAinhibitory against beta-2 microglobulin, wherein the shRNA has asequence comprising any one of SEQ ID NOs: 2-4. In particularembodiments, the shRNA has a sequence comprising SEQ ID NO: 2. In somesuch embodiments, the first expression cassette is in a 3′ to 5′orientation and the second expression cassette is in a 5′ to 3′orientation relative to the 5′ and 3′ homology arms, and the firstexpression cassette is 5′ upstream of the second expression cassette. Insome such embodiments, the first expression cassette comprises: (i) anucleic acid sequence encoding a chimeric antigen receptor or anexogenous T cell receptor; (ii) a JeT promoter which drives expressionof the chimeric antigen receptor or the exogenous T cell receptor; and(iii) a polyA sequence; and the second expression cassette comprises:(iv) a nucleic acid sequence encoding the shRNA; (v) a U6 promoter whichdrives expression of the shRNA; and (vi) a central polypurine tract andcentral terminator sequence (cPPT/CTS) sequence. In some suchembodiments, the nucleic acid molecule comprises a first copy and asecond copy of the second expression cassette, wherein the first copyand the second copy are identical, and wherein the first copy and thesecond copy are in tandem, and further wherein the first copy and thesecond copy are in the same orientation.

In some embodiments, the nucleic acid sequence encoding the engineeredantigen receptor is integrated at the same location within the genome asthe nucleic acid sequence encoding the inhibitory nucleic acid molecule.In particular embodiments, the genetically-modified eukaryotic cellcomprises in its genome the nucleic acid molecule of the invention.

In other embodiments, the nucleic acid sequence encoding the engineeredantigen receptor is integrated at a different location within the genomethan the nucleic acid sequence encoding the inhibitory nucleic acidmolecule.

In some embodiments, the genetically-modified eukaryotic cell is lesssusceptible to endogenous NK cell killing when compared to a controlcell, has extended persistence in a subject when compared to a controlcell, exhibits enhanced expansion in a subject when compared to acontrol cell, and/or exhibits reduced allogenicity when compared to acontrol cell.

In some embodiments, the engineered antigen receptor is a chimericantigen receptor or an exogenous T cell receptor.

In some embodiments, the genetically-modified eukaryotic cell is agenetically-modified human T cell.

In particular embodiments, the genetically-modified eukaryotic cell is agenetically-modified human T cell, and the engineered antigen receptoris a chimeric antigen receptor or an exogenous T cell receptor.

In another aspect, the invention provides a pharmaceutical compositioncomprising a pharmaceutically-acceptable carrier and a therapeuticallyeffective amount of any genetically-modified eukaryotic cell describedherein above.

In some particular embodiments, the genetically-modified eukaryotic cellof the pharmaceutical composition is a genetically-modified human Tcell, the engineered antigen receptor is a chimeric antigen receptor orexogenous T cell receptor, and cell surface expression of beta-2microglobulin is between about 1% and about 50%, about 1% and about 25%,about 1% and about 10%, or about 1% and about 5% of cell surface beta-2microglobulin expression on a control cell.

In other particular embodiments, the genetically-modified eukaryoticcell of the pharmaceutical composition is a genetically-modified human Tcell, the engineered antigen receptor is a chimeric antigen receptor orexogenous T cell receptor, and cell surface expression of beta-2microglobulin on the genetically-modified human T cell is reduced by 10%to 95%, by 50% to 95%, by 75% to 95%, or by 90% to 95% compared to cellsurface expression of beta-2 microglobulin on a control cell.

In some particular embodiments, the genetically-modified eukaryotic cellof the pharmaceutical composition is a genetically-modified human Tcell, the engineered antigen receptor is a chimeric antigen receptor orexogenous T cell receptor, and cell surface expression of MHC class Imolecules is between about 1% and about 50%, about 1% and about 25%,about 1% and about 10%, or about 1% and about 5% of cell surfaceexpression of MHC class I molecules on a control cell.

In other particular embodiments, the genetically-modified eukaryoticcell of the pharmaceutical composition is a genetically-modified human Tcell, the engineered antigen receptor is a chimeric antigen receptor orexogenous T cell receptor, and cell surface expression of MHC class Imolecules on the genetically-modified human T cell is reduced by 10% to95%, by 50% to 95%, by 75% to 95%, or by 90% to 95% compared to cellsurface expression of MHC class I molecules on a control cell.

In other particular embodiments, the genetically-modified eukaryoticcell of the pharmaceutical composition is a genetically-modified human Tcell, and the engineered antigen receptor is a chimeric antigenreceptor, and cell surface expression of CD52 is between about 1% andabout 50%, about 1% and about 25%, about 1% and about 10%, or about 1%and about 5% of cell surface CD52 expression on a control cell.

In some embodiments, the genetically-modified eukaryotic cell of thepharmaceutical composition is a genetically-modified human T cell, theengineered antigen receptor is a chimeric antigen receptor or exogenousT cell receptor, and cell surface expression of CD52 on thegenetically-modified human T cell is reduced by 10% to 95%, by 50% to95%, by 75% to 95%, or by 90% to 95% compared to cell surface expressionof CD52 on a control cell.

In certain embodiments, the pharmaceutical composition of the inventionis for immunotherapy in the treatment of cancer in a subject in needthereof.

In another aspect, the invention provides a population ofgenetically-modified eukaryotic cells comprising a plurality of anygenetically-modified eukaryotic cell described herein.

In some embodiments, at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or up to 100%, of cellsin the population are a genetically-modified eukaryotic cell asdescribed herein.

In particular embodiments, the genetically-modified eukaryotic cells ofthe population are genetically-modified human T cells, or cells derivedtherefrom, or genetically-modified NK cells, or cells derived therefrom.

In certain embodiments, the genetically-modified eukaryotic cells of thepopulation comprise a cell surface chimeric antigen receptor orexogenous T cell receptor. In some of these embodiments, the chimericantigen receptor or exogenous T cell receptor comprises an extracellularligand-binding domain having specificity for a tumor-specific antigen.

In some embodiments, the genetically-modified eukaryotic cells of thepopulation have no cell surface expression of an endogenous T cellreceptor when compared to an unmodified control cell. In someembodiments, the genetically-modified eukaryotic cells of the populationhave reduced cell surface expression of beta-2 microglobulin, MEW classI molecules, or CD52.

In another aspect, the invention provides a method of usingimmunotherapy to treat a disease in a subject in need thereof, themethod comprising administering to the subject a genetically-modifiedeukaryotic cell described herein; wherein the genetically-modifiedeukaryotic cell is a genetically-modified human T cell expressing achimeric antigen receptor or an exogenous T cell receptor; and whereincell surface expression of beta-2 microglobulin on thegenetically-modified human T cell is between about 1% and about 50%,about 1% and about 25%, about 1% and about 10%, or about 1% and about 5%of cell surface beta-2 microglobulin expression on a control cell.

In some embodiments of the method, cell surface expression of beta-2microglobulin on the genetically-modified human T cell is reduced by 10%to 95%, by 50% to 95%, by 75% to 95%, or by 90% to 95% compared to cellsurface beta-2 microglobulin expression on a control cell.

In some embodiments of the method, endogenous NK cell killing of thegenetically-modified human T cell is reduced in the subject whencompared to a genetically-modified human T cell having no cell surfacebeta-2 microglobulin expression.

In some embodiments of the method, the subject is administered anypharmaceutical composition described herein in which cell surface beta-2microglobulin expression is reduced on the genetically-modified human Tcell when compared to a control cell.

In some embodiments of the method, the genetically-modified human T cellis allogeneic to the subject.

In some embodiments of the method, persistence of thegenetically-modified human T cell is extended in the subject whencompared to a genetically-modified human T cell having no cell surfacebeta-2 microglobulin expression, or when compared to agenetically-modified human T cell having a wild-type level of cellsurface expression of beta-2 microglobulin.

In some embodiments of the method, expansion of the genetically-modifiedhuman T cell is enhanced in the subject when compared to agenetically-modified human T cell having no cell surface beta-2microglobulin expression, or when compared to a genetically-modifiedhuman T cell having a wild-type level of cell surface expression ofbeta-2 microglobulin.

In some embodiments of the method, allogenicity of thegenetically-modified human T cell is reduced when compared to agenetically-modified human T cell having a wild-type level of cellsurface expression of beta-2 microglobulin.

In some embodiments of the method, the disease is cancer.

In some embodiments of the method, the cancer is selected from the groupconsisting of a cancer of carcinoma, lymphoma, sarcoma, blastomas, andleukemia. In certain embodiments of the method, the cancer is selectedfrom the group consisting of a cancer of B-cell origin, breast cancer,gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma,prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer,rhabdomyo sarcoma, leukemia, and Hodgkin's lymphoma. In particularembodiments of the method, the cancer of B-cell origin is selected fromthe group consisting of B-lineage acute lymphoblastic leukemia, B-cellchronic lymphocytic leukemia, and B-cell non-Hodgkin's lymphoma.

In another aspect, the invention provides a method of usingimmunotherapy to treat a disease in a subject in need thereof, themethod comprising administering to the subject a genetically-modifiedeukaryotic cell described herein; wherein the genetically-modifiedeukaryotic cell is a genetically-modified human T cell expressing achimeric antigen receptor or an exogenous T cell receptor; and whereincell surface expression of MHC class I molecules on thegenetically-modified human T cell is between about 1% and about 50%,about 1% and about 25%, about 1% and about 10%, or about 1% and about 5%of cell surface expression of MHC class I molecules on a control cell.

In some embodiments of the method, cell surface expression of MHC classI molecules on the genetically-modified human T cell is reduced by 10%to 95%, by 50% to 95%, by 75% to 95%, or by 90% to 95% compared to cellsurface expression of MHC class I molecules on a control cell.

In some embodiments of the method, endogenous NK cell killing of thegenetically-modified human T cell is reduced in the subject whencompared to a genetically-modified human T cell having no cell surfaceexpression of MHC class I molecules.

In some embodiments of the method, the subject is administered anypharmaceutical composition described herein in which cell surfaceexpression of MHC class I molecules is reduced on thegenetically-modified human T cell when compared to a control cell.

In some embodiments of the method, the genetically-modified human T cellis allogeneic to the subject.

In some embodiments of the method, persistence of thegenetically-modified human T cell is extended in the subject whencompared to a genetically-modified human T cell having no cell surfaceMHC class I molecule expression, or when compared to agenetically-modified human T cell having a wild-type level of cellsurface expression of MHC class I molecules.

In some embodiments of the method, expansion of the genetically-modifiedhuman T cell is enhanced in the subject when compared to agenetically-modified human T cell having no cell surface MHC class Imolecule expression, or when compared to a genetically-modified human Tcell having a wild-type level of cell surface expression of MHC class Imolecules.

In some embodiments of the method, allogenicity of thegenetically-modified human T cell is reduced when compared to agenetically-modified human T cell having a wild-type level of cellsurface expression of MHC class I molecules.

In some embodiments of the method, the disease is cancer.

In some embodiments of the method, the cancer is selected from the groupconsisting of a cancer of carcinoma, lymphoma, sarcoma, blastomas, andleukemia. In certain embodiments of the method, the cancer is selectedfrom the group consisting of a cancer of B-cell origin, breast cancer,gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma,prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer,rhabdomyo sarcoma, leukemia, and Hodgkin's lymphoma. In particularembodiments of the method, the cancer of B-cell origin is selected fromthe group consisting of B-lineage acute lymphoblastic leukemia, B-cellchronic lymphocytic leukemia, and B-cell non-Hodgkin's lymphoma.

In another aspect, the invention provides a method of usingimmunotherapy to treat cancer in a subject in need thereof, the methodcomprising administering to the subject a genetically-modifiedeukaryotic cell described herein; wherein the genetically-modifiedeukaryotic cell is a genetically-modified human T cell expressing achimeric antigen receptor or an exogenous T cell receptor; and whereincell surface expression of CD52 on the genetically-modified human T cellis between 1% and 50%, 1% and 25%, 1% and 10%, or 1% and 5% of cellsurface CD52 expression on a control cell.

In some embodiments of the method, cell surface expression of CD52 onthe genetically-modified human T cell is reduced by 10% to 95%, by 50%to 95%, by 75% to 95%, or by 90% to 95% compared to cell surfaceexpression of CD52 on a control cell.

In some embodiments of the method, the subject is administered apharmaceutical composition described herein in which cell surfaceexpression of CD52 is reduced on the genetically-modified human T cellwhen compared to a control cell.

In some embodiments of the method, the genetically-modified human T cellis allogeneic to the subject.

In some embodiments of the method, the cancer is selected from the groupconsisting of a cancer of carcinoma, lymphoma, sarcoma, blastomas, andleukemia. In certain embodiments of the method, the cancer is selectedfrom the group consisting of a cancer of B-cell origin, breast cancer,gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma,prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer,rhabdomyo sarcoma, leukemia, and Hodgkin's lymphoma. In particularembodiments of the method, the cancer of B-cell origin is selected fromthe group consisting of B-lineage acute lymphoblastic leukemia, B-cellchronic lymphocytic leukemia, and B-cell non-Hodgkin's lymphoma.

In another aspect, the invention provides a method for preparing anenriched population of genetically-modified eukaryotic cells comprisingan engineered antigen receptor, the method comprising preparing apopulation of cells comprising a genetically-modified eukaryotic celldescribed herein and cells expressing a wild-type level of cell surfaceCD52, wherein cell surface expression of CD52 on thegenetically-modified eukaryotic cell is between about 1% and about 50%,about 1% and about 25%, about 1% and about 10%, or about 1% and about 5%when compared to a control cell, the method comprising: (a) contactingthe population of cells with beads conjugated to an anti-CD52 bindingmolecule, wherein cells expressing a wild-type level of cell surfaceCD52 are bound to the beads and the genetically-modified eukaryotic cellis not bound to the beads; and (b) removing the beads from thepopulation of cells to produce the enriched population of cells; whereinthe enriched population of cells is enriched for thegenetically-modified eukaryotic cell.

In some embodiments of the method, cell surface expression of CD52 onthe genetically-modified eukaryotic cell is reduced by 10% to 95%, by50% to 95%, by 75% to 95%, or by 90% to 95% when compared to a controlcell.

In some embodiments of the method, the beads are magnetic beads. Incertain embodiments of the method, the magnetic beads are removed fromthe population of cells by magnetic separation.

In some embodiments of the method, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, atleast about 95%, or up to 100% of cells in the enriched population arethe genetically-modified eukaryotic cell.

In some embodiments of the method, the genetically-modified eukaryoticcell expresses a chimeric antigen receptor. In other embodiments of themethod, the genetically-modified eukaryotic cell expresses an exogenousT cell receptor.

In some embodiments of the method, the genetically-modified eukaryoticcell is a genetically-modified human T cell, such as anygenetically-modified T cell described herein.

In another aspect, the present disclosure provides agenetically-modified eukaryotic cell described herein for use as amedicament. The present disclosure further provides the use of agenetically-modified eukaryotic cell described herein in the manufactureof a medicament for treating a disease in a subject in need thereof. Inone such embodiment, the medicament is useful in the treatment ofcancer.

The foregoing and other aspects and embodiments of the present inventioncan be more fully understood by reference to the following detaileddescription and claims. Certain features of the invention, which are,for clarity, described in the context of separate embodiments, may alsobe provided in combination in a single embodiment. All combinations ofthe embodiments are specifically embraced by the present invention andare disclosed herein just as if each and every combination wasindividually and explicitly disclosed. Conversely, various features ofthe invention, which are, for brevity, described in the context of asingle embodiment, may also be provided separately or in any suitablesub-combination. All sub-combinations of features listed in theembodiments are also specifically embraced by the present invention andare disclosed herein just as if each and every such sub-combination wasindividually and explicitly disclosed herein. Embodiments of each aspectof the present invention disclosed herein apply to each other aspect ofthe invention mutatis mutandis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows diagrams of various embodiments of the nucleic acidmolecule of the invention. The JeT promoter is shown as an example of apromoter driving expression of the engineered antigen receptor. A U6promoter is shown as an example of a promoter driving expression of theinhibitory nucleic acid molecule. A chimeric antigen receptor (CAR) isshown as an example of an engineered antigen receptor. An shRNA is shownas an example of an inhibitory nucleic acid molecule. A poly-A sequenceis shown as an example of a sequence which terminates translation of theengineered antigen receptor. A cPPT/CTS sequence is shown as an exampleof a sequence which terminates translation of the inhibitory nucleicacid molecule. 5′ and 3′ homology arms are shown flanking the firstexpression cassette and second expression cassette of each construct.Optional 5′ and 3′ inverted terminal repeats are further shown in eachconstruct. Constructs above the dashed line have first and secondexpression cassettes in the same orientation, whereas constructs belowthe dashed line have first and second expression cassettes in oppositeorientations.

FIG. 2A-2L shows flow plots which represent NK cell killing of primaryhuman T cells. The indicated ratios represent the ratio of NK cells to Tcells (E:T) in each experiment. FIG. 2A shows NK cell killing of B2M+ Tcells using a 2:1 ratio. FIG. 2B shows NK cell killing of B2M+ T cellsusing a 1:1 ratio. FIG. 2C shows NK cell killing of B2M− T cells using a0.5:1 ratio. FIG. 2D shows NK cell killing of B2M− T cells using a 0:1ratio. FIG. 2E shows NK cell killing of B2M- T cells using a 2:1 ratio.FIG. 2F shows NK cell killing of B2M- T cells using a 1:1 ratio. FIG. 2Gshows NK cell killing of B2M+ T cells using a 0.5:1 ratio. FIG. 2H showsNK cell killing of B2M− T cells using a 0:1 ratio. FIG. 2I shows NK cellkilling of Daudi Class I-negative cells using a 2:1 ratio. FIG. 2J showsNK cell killing of Daudi Class I-negative cells using a 1:1 ratio. FIG.2K shows NK cell killing of Daudi Class I-negative cells using a 0.5:1ratio. FIG. 2L shows NK cell killing of Daudi Class I-negative cellsusing a 0:1 ratio.

FIG. 3 shows a chart summarizing NK cell killing of B2M+ and B2M− cellsat different ratios.

FIG. 4 shows percentage knockdown of human B2M in primary human T cellsby three candidate B2M shRNAs.

FIGS. 5A-5D show flow diagrams representing NK cell lysis or allogeneiccell lysis of K562 cells or mock-treated primary human T cells. FIG. 5Ashows NK cell lysis of K562 cells. FIG. 5B shows allogeneic cell lysisof K562 cells. FIG. 5C shows NK cell lysis of mock-treated primary humanT cells. FIG. 5D shows allogeneic cell lysis of mock-treated primaryhuman T cells.

FIGS. 6A-6D show flow diagrams representing NK cell lysis or allogeneiccell lysis of primary human T cells treated with B2M shRNAs. FIG. 6Ashows NK cell lysis of primary human T cells treated with shRNA254. FIG.6B shows allogeneic cell lysis of primary human T cells treated withshRNA254. FIG. 6C shows NK cell lysis of primary human T cells treatedwith shRNA472. FIG. 6D shows allogeneic cell lysis of primary human Tcells treated with shRNA472.

FIGS. 7A-7F show diagrams of various nucleic acid molecule constructsencoding a chimeric antigen receptor and an shRNA against beta-2microglobulin. FIG. 7A shows construct 7007 (SEQ ID NO: 18). FIG. 7Bshows construct 7217 (SEQ ID NO: 19). FIG. 7C shows construct 7008 (SEQID NO: 20). FIG. 7D shows construct 7218 (SEQ ID NO: 21). FIG. 7E showsconstruct 7009 (SEQ ID NO: 22). FIG. 7F shows construct 7219 (SEQ ID NO:23).

FIG. 8 shows percentage knockdown of human CD52 in primary human T cellsby three different candidate CD52 shRNAs.

FIG. 9 A-C show knockdown of CD52 using shRNA and magnetic enrichment ofthe knockdown population of primary human T cells by CD52 magneticdepletion. FIG. 9A shows T cells that were mock transduced. FIG. 9Bshows T cells transduced with an shRNA-568 lentivirus. FIG. 9C showslentivirus-shRNA568 transduced cells that have undergone a CD52 magneticdepletion.

FIGS. 10A-10H shows diagrams of nucleic acid molecule constructsencoding a chimeric antigen receptor and an shRNA against CD52. FIG. 10Ashows construct 7005 (SEQ ID NO: 10) which encodes a CAR only. FIG. 10Bshows construct 7002 (SEQ ID NO: 11) which encodes a CAR only. FIG. 10Cshows construct 7004 (SEQ ID NO: 12). FIG. 10D shows construct 7204 (SEQID NO: 13). FIG. 10E shows construct 7013 (SEQ ID NO: 14). FIG. 10Fshows construct 7213 (SEQ ID NO: 15). FIG. 10G shows construct 7014 (SEQID NO: 16). FIG. 10H shows construct 7214 (SEQ ID NO: 17).

FIG. 11A-11D shows CD52 knockdown profiles using CAR/CD52 shRNAconstructs with different orientations. FIG. 11A shows CD52 expressionwhen a CAR is expressed in the absence of a CD52 shRNA. FIG. 11B showsCD52 expression when using the 7013 construct. FIG. 11C shows CD52expression when using the 7004 construct. FIG. 11D shows CD52 expressionwhen using the 7014 construct.

FIG. 12A-12C shows B2M knockdown on CAR T cells using CAR/B2M shRNAconstructs having one or multiple shRNA cassettes. FIG. 12A shows B2Mexpression in CAR T cells expressing no B2M shRNA (7002—shaded curve) ora single B2M shRNA cassette (7008—open curve). FIG. 12B shows B2Mexpression in CAR T cells expressing no B2M shRNA (7002—shaded) or twoB2M shRNA cassettes (7029-open). FIG. 12C shows B2M expression inCAR-/CD3+(i.e. non-edited) populations from cultures electroporated with7002, 7008, or 7029.

FIG. 13A-13C shows cell surface expression of beta-2 microglobulin on Tcells transfected with linearized DNA to express a control CAR-negativeconstruct (7002), CAR constructs expressing a single shRNA472 copy in a3′ to 5′ head-to-tail configuration with the CAR (7056), or in a 3′ to5′/5′ to 3′ tail-to-tail configuration with the CAR (7059), or a CARconstruct expressing two shRNA cassette copies in a 3′ to 5′/5′ to 3′tail-to-tail configuration with the CAR (7060). FIG. 13A shows CAR Tcells expressing the 7002 and 7056 constructs. FIG. 13B shows CAR Tcells expressing the 7002 and 7059 constructs. FIG. 13C shows CAR Tcells expressing the 7002 and 7060 constructs.

FIG. 14 A-D shows beta-2 microglobulin expression or HLA-A, B, and Cexpression (i.e., MHC class I molecule expression) on T cells transducedwith an AAV comprising construct 7056 which expresses a single copy ofthe shRNA472 in a 3′ to 5′ head-to-tail configuration with the CAR. FIG.14A shows the B2M surface levels in CD3−/CAR+ cells compared tomeganuclease-edited cells expressing no shRNA from a control culture.FIG. 14B shows B2M levels on CD3−/CAR+ versus CD3+/CAR− populations inthe same culture. FIG. 14C shows HLA-ABC (i.e., MHC class I molecule)surface levels in CD3−/CAR+ cells compared to meganuclease-edited cellsexpressing no shRNA from a control culture. FIG. 14D shows HLA-ABClevels on CD3−/CAR+ versus CD3+/CAR− populations in the same culture.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 sets forth the nucleic acid sequence of the TRC 1-2recognition sequence.

SEQ ID NO: 2 sets forth the nucleic acid sequence of the anti-beta-2microglobulin shRNA472.

SEQ ID NO: 3 sets forth the nucleic acid sequence of the anti-beta-2microglobulin shRNA256.

SEQ ID NO: 4 sets forth the nucleic acid sequence of the anti-beta-2microglobulin shRNA254.

SEQ ID NO: 5 sets forth the nucleic acid sequence of the anti-CD52shRNA572.

SEQ ID NO: 6 sets forth the nucleic acid sequence of the anti-CD52shRNA876.

SEQ ID NO: 7 sets forth the nucleic acid sequence of the anti-CD52shRNA568.

SEQ ID NO: 8 sets forth the nucleic acid sequence of the anti-CD52shRNA569.

SEQ ID NO: 9 sets forth the nucleic acid sequence of the anti-CD52shRNA571.

SEQ ID NO: 10 sets forth the nucleic acid sequence of the CAR 7005construct.

SEQ ID NO: 11 sets forth the nucleic acid sequence of the CAR 7002construct.

SEQ ID NO: 12 sets forth the nucleic acid sequence of the CAR 7004construct.

SEQ ID NO: 13 sets forth the nucleic acid sequence of the CAR 7204construct.

SEQ ID NO: 14 sets forth the nucleic acid sequence of the CAR 7013construct.

SEQ ID NO: 15 sets forth the nucleic acid sequence of the CAR 7213construct.

SEQ ID NO: 16 sets forth the nucleic acid sequence of the CAR 7014construct.

SEQ ID NO: 17 sets forth the nucleic acid sequence of the CAR 7214construct.

SEQ ID NO: 18 sets forth the nucleic acid sequence of the CAR 7007construct.

SEQ ID NO: 19 sets forth the nucleic acid sequence of the CAR 7217construct.

SEQ ID NO: 20 sets forth the nucleic acid sequence of the CAR 7008construct.

SEQ ID NO: 21 sets forth the nucleic acid sequence of the CAR 7218construct.

SEQ ID NO: 22 sets forth the nucleic acid sequence of the CAR 7009construct.

SEQ ID NO: 23 sets forth the nucleic acid sequence of the CAR 7219construct.

SEQ ID NO: 24 sets forth the nucleic acid sequence of the CAR 7029construct.

SEQ ID NO: 25 sets forth the nucleic acid sequence of the CAR 7056construct.

SEQ ID NO: 26 sets forth the nucleic acid sequence of the CAR 7059construct.

SEQ ID NO: 27 sets forth the nucleic acid sequence of the CAR 7060construct.

SEQ ID NO: 28 sets forth the nucleic acid sequence of the cPPT/CTSsequence.

DETAILED DESCRIPTION OF THE INVENTION 1.1 References and Definitions

The patent and scientific literature referred to herein establishesknowledge that is available to those of skill in the art. The issued USpatents, allowed applications, published foreign applications, andreferences, including GenBank database sequences, which are cited hereinare hereby incorporated by reference to the same extent as if each wasspecifically and individually indicated to be incorporated by reference.

The present disclosure can be embodied in different forms and should notbe construed as limited to the embodiments set forth herein. Rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey the scope of the present disclosureto those skilled in the art. For example, features illustrated withrespect to one embodiment can be incorporated into other embodiments,and features illustrated with respect to a particular embodiment can bedeleted from that embodiment. In addition, numerous variations andadditions to the embodiments suggested herein will be apparent to thoseskilled in the art in light of the instant disclosure, which do notdepart from the present disclosure.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. The terminology used in thedescription of the present disclosure herein is for the purpose ofdescribing particular embodiments only and is not intended to belimiting of the present disclosure.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference herein in their entirety.

As used herein, “a,” “an,” or “the” can mean one or more than one. Forexample, “a” cell can mean a single cell or a multiplicity of cells.

As used herein, unless specifically indicated otherwise, the word “or”is used in the inclusive sense of “and/or” and not the exclusive senseof “either/or.”

The terms “expression cassette,” “recombinant DNA construct,”“recombinant construct,” “expression construct,” “chimeric construct,”“construct,” and “recombinant DNA fragment” are used interchangeablyherein and are nucleic acid fragments. A recombinant construct comprisesan artificial combination of nucleic acid fragments, including, withoutlimitation, regulatory and coding sequences that are not found togetherin nature. For example, a recombinant DNA construct may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source and arranged in a manner different than that foundin nature. Such a construct may be used by itself or may be used inconjunction with a vector.

As used herein, a “vector” or “recombinant DNA vector” may be aconstruct that includes a replication system and sequences that arecapable of transcription and translation of a polypeptide-encodingsequence in a given host cell. If a vector is used then the choice ofvector is dependent upon the method that will be used to transform hostcells as is well known to those skilled in the art. Vectors can include,without limitation, plasmid vectors and recombinant lentiviral orrecombinant AAV vectors, or any other vector known in that art suitablefor delivering a gene encoding a co-stimulatory domain of the presentdisclosure to a target cell. The skilled artisan is well aware of thegenetic elements that must be present on the vector in order tosuccessfully transform, select and propagate host cells comprising anyof the isolated nucleotides or nucleic acid sequences of the presentdisclosure.

As used herein, a “vector” can also refer to a viral vector. Viralvectors can include, without limitation, retroviral vectors, lentiviralvectors, adenoviral vectors, and adeno-associated viral vectors (AAV).

As used herein, the term “operably linked” is intended to mean afunctional linkage between two or more elements. For example, anoperable linkage between a nucleic acid sequence encoding a nuclease asdisclosed herein and a regulatory sequence (e.g., a promoter) is afunctional link that allows for expression of the nucleic acid sequenceencoding the nuclease. Operably linked elements may be contiguous ornon-contiguous. When used to refer to the joining of two protein codingregions, by operably linked is intended that the coding regions are inthe same reading frame.

As used herein, the term “RNA interference” or “RNAi” refers to aphenomenon in which the introduction of double-stranded RNA (dsRNA) intoa diverse range of organisms and cell types causes degradation of thecomplementary mRNA. In the cell, long dsRNAs are cleaved into short21-25 nucleotide small interfering RNAs, or siRNAs, by a ribonucleaseknown as Dicer. The siRNAs subsequently assemble with protein componentsinto an RNA-induced silencing complex (RISC), unwinding in the process.Activated RISC then binds to complementary transcript by base pairinginteractions between the siRNA antisense strand and the mRNA. The boundmRNA is cleaved and sequence specific degradation of mRNA results ingene silencing. See, for example, U.S. Pat. No. 6,506,559.

The term “siRNA” as used herein refers to small interfering RNA, alsoknown as short interfering RNA or silencing RNA. siRNAs can be, forexample, 18 to 30, 20 to 25, 21 to 23 or 21 nucleotide-longdouble-stranded RNA molecules. An “shRNA” as used herein is a shorthairpin RNA, which is a sequence of RNA that makes a tight hairpin turnthat can also be used to silence gene expression via RNA interference.shRNA can by operably linked to the U6 promoter expression. The shRNAhairpin structure is cleaved by the cellular machinery into siRNA. shRNAdisclosed herein can comprise a sequence complementary to at least 13nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, atleast 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides,at least 22 nucleotides, or 23 nucleotides of the mRNA a target protein.

As used herein, an “engineered antigen receptor” refers to an exogenousreceptor introduced into a cell, such as a chimeric antigen receptor orexogenous T cell receptor, which induces an activating signal in thecell upon stimulation/binding to a ligand or antigen (e.g., atumor-specific antigen).

As used herein, a “chimeric antigen receptor” or “CAR” refers to anengineered receptor that grafts specificity for an antigen or otherligand or molecule onto an immune effector cell (e.g., a T cell or NKcell). A chimeric antigen receptor typically comprises at least anextracellular ligand-binding domain or moiety and an intracellulardomain that comprises one or more signaling domains and/orco-stimulatory domains.

In some embodiments, the extracellular ligand-binding domain or moietyis in the form of a single-chain variable fragment (scFv) derived from amonoclonal antibody, which provides specificity for a particular epitopeor antigen (e.g., an epitope or antigen preferentially present on thesurface of a cell, such as a cancer cell or other disease-causing cellor particle). In some embodiments, the scFv is attached via a linkersequence. In some embodiments, the extracellular ligand-binding domainis specific for any antigen or epitope of interest. In some embodiments,the scFv is humanized. In some embodiments, the extracellular domain ofa chimeric antigen receptor comprises an autoantigen (see, Payne et al.(2016) Science, Vol. 353 (6295): 179-184), which is recognized byautoantigen-specific B cell receptors on B lymphocytes, thus directing Tcells to specifically target and kill autoreactive B lymphocytes inantibody-mediated autoimmune diseases. Such CARs can be referred to aschimeric autoantibody receptors (CAARs), and the incorporation of one ormore co-stimulatory domains described herein into such CAARs isencompassed by the present disclosure.

The extracellular domain of a chimeric antigen receptor can alsocomprise a naturally-occurring ligand for an antigen of interest, or afragment of a naturally-occurring ligand which retains the ability tobind the antigen of interest.

Intracellular signaling domains are cytoplasmic domains which transmitan activation signal to the cell following binding of the extracellulardomain. An intracellular signaling domain can be any intracellularsignaling domain of interest that is known in the art. Such cytoplasmicsignaling domains can include, without limitation, CD3 ξ.

In some embodiments, the intracellular domain also includes one or moreintracellular co-stimulatory domains, such as those described herein,which transmit a co-stimulatory signal which promotes cellproliferation, cell survival, and/or cytokine secretion after binding ofthe extracellular domain. As used herein, a “co-stimulatory domain”refers to a polypeptide domain which transmits an intracellularproliferative and/or cell-survival signal upon activation. Activation ofa co-stimulatory domain may occur following homodimerization of twoco-stimulatory domain polypeptides. Activation may also occur, forexample, following activation of a construct comprising theco-stimulatory domain (e.g., a chimeric antigen receptor or an inducibleregulatory construct). Generally, a co-stimulatory domain can be derivedfrom a transmembrane co-stimulatory receptor, particularly from anintracellular portion of a co-stimulatory receptor. Such intracellularco-stimulatory domains can be any of those known in the art and caninclude, without limitation, CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30,CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2,CD7, LIGHT, NKG2C, B7-H3 and a ligand that specifically binds with CD83,N1, N6, or any combination thereof.

As used herein, a “co-stimulatory signal” refers to an intracellularsignal induced by a co-stimulatory domain that promotes cellproliferation, expansion of a cell population in vitro and/or in vivo,promotes cell survival, modulates (e.g., upregulates or downregulates)the secretion of cytokines, and/or modulates the production and/orsecretion of other immunomodulatory molecules. In some embodiments, aco-stimulatory signal is induced following homodimerization of twoco-stimulatory domain polypeptides. In some embodiments, aco-stimulatory signal is induced following activation of a constructcomprising the co-stimulatory domain (e.g., a chimeric antigen receptoror an inducible regulatory construct).

A chimeric antigen receptor can further include additional structuralelements, including a transmembrane domain that is attached to theextracellular ligand-binding domain via a hinge or spacer sequence. Thetransmembrane domain can be derived from any membrane-bound ortransmembrane protein. For example, the transmembrane polypeptide can bea subunit of the T-cell receptor (i.e., an α, β, γ or ξ, polypeptideconstituting CD3 complex), IL2 receptor p55 (a chain), p75 (β chain) ory chain, subunit chain of Fc receptors (e.g., Fcy receptor III) or CDproteins such as the CD8 alpha chain. Alternatively the transmembranedomain can be synthetic and can comprise predominantly hydrophobicresidues such as leucine and valine.

The hinge region refers to any oligo- or polypeptide that functions tolink the transmembrane domain to the extracellular ligand-bindingdomain. For example, a hinge region may comprise up to 300 amino acids,preferably 10 to 100 amino acids and most preferably 25 to 50 aminoacids. Hinge regions may be derived from all or part of naturallyoccurring molecules, such as from all or part of the extracellularregion of CD8, CD4 or CD28, or from all or part of an antibody constantregion. Alternatively, the hinge region may be a synthetic sequence thatcorresponds to a naturally occurring hinge sequence, or may be anentirely synthetic hinge sequence. In particular examples, a hingedomain can comprise a part of a human CD8 alpha chain, FcyRIIIa receptoror IgGl.

As used herein, the term “activation” refers to the state of a cell(e.g., a T cell) that has been sufficiently stimulated to inducedetectable effector function. In some embodiments, activation isassociated with induced cytokine production and/or induced cellproliferation and expansion.

As used herein, an “exogenous T cell receptor” or “exogenous TCR” refersto a TCR whose sequence is introduced into the genome of an immuneeffector cell (e.g., a human T cell) that may or may not endogenouslyexpress the TCR. Expression of an exogenous TCR on an immune effectorcell can confer specificity for a specific epitope or antigen (e.g., anepitope or antigen preferentially present on the surface of a cancercell or other disease-causing cell or particle). Such exogenous T cellreceptors can comprise alpha and beta chains or, alternatively, maycomprise gamma and delta chains. Exogenous TCRs useful in the inventionmay have specificity to any antigen or epitope of interest.

As used herein, with respect to a protein, the term “engineered” or“recombinant” means having an altered amino acid sequence as a result ofthe application of genetic engineering techniques to nucleic acids whichencode the protein, and cells or organisms which express the protein.With respect to a nucleic acid, the term “recombinant” means having analtered nucleic acid sequence as a result of the application of geneticengineering techniques. Genetic engineering techniques include, but arenot limited to, PCR and DNA cloning technologies; transfection,transformation and other gene transfer technologies; homologousrecombination; site-directed mutagenesis; and gene fusion. In accordancewith this definition, a protein having an amino acid sequence identicalto a naturally-occurring protein, but produced by cloning and expressionin a heterologous host, is not considered recombinant.

As used herein, the term “wild-type” refers to the most common naturallyoccurring polynucleotide or polypeptide sequence responsible for a givenphenotype. Whereas a wild-type allele or polypeptide can confer a normalphenotype in an organism, a mutant or variant allele or polypeptide can,in some instances, confer an altered phenotype.

As used herein with respect to recombinant proteins, the term“modification” means any insertion, deletion or substitution of an aminoacid residue in the recombinant sequence relative to a referencesequence (e.g., a wild-type or a native sequence).

As used herein, the term “meganuclease” refers to an endonuclease thatbinds double-stranded DNA at a recognition sequence that is greater than12 base pairs. In some embodiments, the recognition sequence for ameganuclease of the present disclosure is 22 base pairs. A meganucleasecan be an endonuclease that is derived from I-CreI, and can refer to anengineered variant of I-CreI that has been modified relative to naturalI-CreI with respect to, for example, DNA-binding specificity, DNAcleavage activity, DNA-binding affinity, or dimerization properties.Methods for producing such modified variants of I-CreI are known in theart (e.g. WO 2007/047859). A meganuclease as used herein binds todouble-stranded DNA as a heterodimer. A meganuclease may also be a“single-chain meganuclease” in which a pair of DNA-binding domains arejoined into a single polypeptide using a peptide linker. The term“homing endonuclease” is synonymous with the term “meganuclease.”Meganucleases of the present disclosure are substantially non-toxic whenexpressed in cells, particularly in human T cells, such that cells canbe transfected and maintained at 37° C. without observing deleteriouseffects on cell viability or significant reductions in meganucleasecleavage activity when measured using the methods described herein.

As used herein, the term “single-chain meganuclease” refers to apolypeptide comprising a pair of nuclease subunits joined by a linker. Asingle-chain meganuclease has the organization: N-terminalsubunit—Linker—C-terminal subunit. The two meganuclease subunits willgenerally be non-identical in amino acid sequence and will recognizenon-identical DNA sequences. Thus, single-chain meganucleases typicallycleave pseudo-palindromic or non-palindromic recognition sequences. Asingle-chain meganuclease may be referred to as a “single-chainheterodimer” or “single-chain heterodimeric meganuclease” although it isnot, in fact, dimeric. For clarity, unless otherwise specified, the term“meganuclease” can refer to a dimeric or single-chain meganuclease.

As used herein, the term “linker” refers to an exogenous peptidesequence used to join two meganuclease subunits into a singlepolypeptide. A linker may have a sequence that is found in naturalproteins, or may be an artificial sequence that is not found in anynatural protein. A linker may be flexible and lacking in secondarystructure or may have a propensity to form a specific three-dimensionalstructure under physiological conditions. A linker can include, withoutlimitation, any of those encompassed by U.S. Pat. Nos. 8,445,251 and9,434,931.

As used herein, the term “zinc finger nuclease” or “ZFN” refers to achimeric protein comprising a zinc finger DNA-binding domain fused to anuclease domain from an endonuclease or exonuclease, including but notlimited to a restriction endonuclease, homing endonuclease, Si nuclease,mung bean nuclease, pancreatic DNAse I, micrococcal nuclease, and yeastHO endonuclease. Nuclease domains useful for the design of zinc fingernucleases include those from a Type IIs restriction endonuclease,including but not limited to FokI, FoM, and StsI restriction enzyme.Additional Type IIs restriction endonucleases are described inInternational Publication No. WO 2007/014275, which is incorporated byreference in its entirety. The structure of a zinc finger domain isstabilized through coordination of a zinc ion. DNA binding proteinscomprising one or more zinc finger domains bind DNA in asequence-specific manner. The zinc finger domain can be a nativesequence or can be redesigned through rational or experimental means toproduce a protein which binds to a pre-determined DNA sequence—18basepairs in length, comprising a pair of nine basepair half-sitesseparated by 2-10 basepairs. See, for example, U.S. Pat. Nos. 5,789,538,5,925,523, 6,007,988, 6,013,453, 6,200,759, and InternationalPublication Nos. WO 95/19431, WO 96/06166, WO 98/53057, WO 98/54311, WO00/27878, WO 01/60970, WO 01/88197, and WO 02/099084, each of which isincorporated by reference in its entirety. By fusing this engineeredprotein domain to a nuclease domain, such as FokI nuclease, it ispossible to target DNA breaks with genome-level specificity. Theselection of target sites, zinc finger proteins and methods for designand construction of zinc finger nucleases are known to those of skill inthe art and are described in detail in U.S. Publications Nos.20030232410, 20050208489, 2005064474, 20050026157, 20060188987 andInternational Publication No. WO 07/014275, each of which isincorporated by reference in its entirety. Cleavage by a zinc fingernuclease can create a blunt end or a 5′ overhand of variable length(frequently four basepairs).

As used herein, the term “TALEN” refers to an endonuclease comprising aDNA-binding domain comprising a plurality of TAL domain repeats fused toa nuclease domain or an active portion thereof from an endonuclease orexonuclease, including but not limited to a restriction endonuclease,homing endonuclease, S1 nuclease, mung bean nuclease, pancreatic DNAseI, micrococcal nuclease, and yeast HO endonuclease. See, for example,Christian et al. (2010) Genetics 186:757-761, which is incorporated byreference in its entirety. Nuclease domains useful for the design ofTALENs include those from a Type IIs restriction endonuclease, includingbut not limited to FokI, FoM, StsI, HhaI, HindIII, Nod, BbvCI, EcoRI,BglI, and AlwI. Additional Type IIs restriction endonucleases aredescribed in International Publication No. WO 2007/014275. In someembodiments, the nuclease domain of the TALEN is a FokI nuclease domainor an active portion thereof. TAL domain repeats can be derived from theTALE (transcription activator-like effector) family of proteins used inthe infection process by plant pathogens of the Xanthomonas genus. TALdomain repeats are 33-34 amino acid sequences with divergent 12th and13th amino acids. These two positions, referred to as the repeatvariable dipeptide (RVD), are highly variable and show a strongcorrelation with specific nucleotide recognition. Each base pair in theDNA target sequence is contacted by a single TAL repeat, with thespecificity resulting from the RVD. In some embodiments, the TALENcomprises 16-22 TAL domain repeats. DNA cleavage by a TALEN requires twoDNA recognition regions flanking a nonspecific central region (i.e., the“spacer”). The term “spacer” in reference to a TALEN refers to thenucleic acid sequence that separates the two nucleic acid sequencesrecognized and bound by each monomer constituting a TALEN. The TALdomain repeats can be native sequences from a naturally-occurring TALEprotein or can be redesigned through rational or experimental means toproduce a protein which binds to a pre-determined DNA sequence (see, forexample, Boch et al. (2009) Science 326(5959):1509-1512 and Moscou andBogdanove (2009) Science 326(5959):1501, each of which is incorporatedby reference in its entirety). See also, U.S. Publication No.20110145940 and International Publication No. WO 2010/079430 for methodsfor engineering a TALEN to recognize a specific sequence and examples ofRVDs and their corresponding target nucleotides. In some embodiments,each nuclease (e.g., FokI) monomer can be fused to a TAL effectorsequence that recognizes a different DNA sequence, and only when the tworecognition sites are in close proximity do the inactive monomers cometogether to create a functional enzyme.

As used herein, the term “compact TALEN” refers to an endonucleasecomprising a DNA-binding domain with one or more TAL domain repeatsfused in any orientation to any portion of the I-TevI homingendonuclease or any of the endonucleases listed in Table 2 in U.S.Application No. 20130117869 (which is incorporated by reference in itsentirety), including but not limited to MmeI EndA, End1, I-BasI,I-TevII, I-TevIII, I-TwoI, MspI, MvaI, NucA, and NucM. Compact TALENs donot require dimerization for DNA processing activity, alleviating theneed for dual target sites with intervening DNA spacers. In someembodiments, the compact TALEN comprises 16-22 TAL domain repeats.

As used herein, the term “CRISPR” refers to a caspase-based endonucleasecomprising a caspase, such as Cas9, and a guide RNA that directs DNAcleavage of the caspase by hybridizing to a recognition site in thegenomic DNA. The caspase component of a CRISPR is an RNA-guided DNAendonuclease. In certain embodiments, the caspase is a class II Casenzyme. In some of these embodiments, the caspase is a class II, type IIenzyme, such as Cas9. In other embodiments, the caspase is a class II,type V enzyme, such as Cpfl. The guide RNA comprises a direct repeat anda guide sequence (often referred to as a spacer in the context of anendogenous CRISPR system), which is complementary to the targetrecognition site. In certain embodiments, the CRISPR further comprises atracrRNA (trans-activating CRISPR RNA) that is complementary (fully orpartially) to a direct repeat sequence (sometimes referred to as atracr-mate sequence) present on the guide RNA. In particularembodiments, the caspase can be mutated with respect to a correspondingwild-type enzyme such that the enzyme lacks the ability to cleave onestrand of a target polynucleotide, functioning as a nickase, cleavingonly a single strand of the target DNA. Non-limiting examples of caspaseenzymes that function as a nickase include Cas9 enzymes with a D10Amutation within the RuvC I catalytic domain, or with a H840A, N854A, orN863A mutation.

As used herein, the term “megaTAL” refers to a single-chain nucleasecomprising a transcription activator-like effector (TALE) DNA bindingdomain with an engineered, sequence-specific homing endonuclease.

As used herein, the term “recognition sequence” refers to a DNA sequencethat is bound and cleaved by an endonuclease. In the case of ameganuclease, a recognition sequence comprises a pair of inverted, 9basepair “half sites” which are separated by four basepairs. In the caseof a single-chain meganuclease, the N-terminal domain of the proteincontacts a first half-site and the C-terminal domain of the proteincontacts a second half-site. Cleavage by a meganuclease produces fourbasepair 3′ “overhangs”. “Overhangs”, or “sticky ends” are short,single-stranded DNA segments that can be produced by endonucleasecleavage of a double-stranded DNA sequence. In the case of meganucleasesand single-chain meganucleases derived from I-CreI, the overhangcomprises bases 10-13 of the 22 basepair recognition sequence. In thecase of a compact TALEN, the recognition sequence comprises a firstCNNNGN sequence that is recognized by the I-Teel domain, followed by anon-specific spacer 4-16 basepairs in length, followed by a secondsequence 16-22 bp in length that is recognized by the TAL-effectordomain (this sequence typically has a 5′ T base). Cleavage by a CompactTALEN produces two basepair 3′ overhangs. In the case of a CRISPR, therecognition sequence is the sequence, typically 16-24 basepairs, towhich the guide RNA binds to direct cleavage. Full complementaritybetween the guide sequence and the recognition sequence is notnecessarily required to effect cleavage. Cleavage by a CRISPR canproduce blunt ends (such as by a class II, type II caspase) oroverhanging ends (such as by a class II, type V caspase), depending onthe caspase. In those embodiments wherein a Cpfl caspase is utilized,cleavage by the CRISPR complex comprising the same will result in 5′overhangs and in certain embodiments, 5 nucleotide 5′ overhangs. Eachcaspase enzyme also requires the recognition of a PAM (protospaceradjacent motif) sequence that is near the recognition sequencecomplementary to the guide RNA. The precise sequence, lengthrequirements for the PAM, and distance from the target sequence differdepending on the caspase enzyme, but PAMs are typically 2-5 base pairsequences adjacent to the target/recognition sequence. PAM sequences forparticular caspase enzymes are known in the art (see, for example, U.S.Pat. No. 8,697,359 and U.S. Publication No. 20160208243, each of whichis incorporated by reference in its entirety) and PAM sequences fornovel or engineered caspase enzymes can be identified using methodsknown in the art, such as a PAM depletion assay (see, for example,Karvelis et al. (2017) Methods 121-122:3-8, which is incorporated hereinin its entirety). In the case of a zinc finger, the DNA binding domainstypically recognize an 18-bp recognition sequence comprising a pair ofnine basepair “half-sites” separated by 2-10 basepairs and cleavage bythe nuclease creates a blunt end or a 5′ overhang of variable length(frequently four basepairs).

As used herein, the term “target site” or “target sequence” refers to aregion of the chromosomal DNA of a cell comprising a recognitionsequence for a nuclease.

As used herein, the term “homologous recombination” or “HR” refers tothe natural, cellular process in which a double-stranded DNA-break isrepaired using a homologous DNA sequence as the repair template (see,e.g. Cahill et al. (2006), Front. Biosci. 11:1958-1976). The homologousDNA sequence may be an endogenous chromosomal sequence or an exogenousnucleic acid that was delivered to the cell.

As used herein, the term “non-homologous end-joining” or “NHEJ” refersto the natural, cellular process in which a double-stranded DNA-break isrepaired by the direct joining of two non-homologous DNA segments (see,e.g. Cahill et al. (2006), Front. Biosci. 11:1958-1976).

As used herein, the term “reduced” refers to any reduction in thesymptoms or severity of a disease or any reduction in the proliferationor number of cancerous cells. In either case, such a reduction may be upto 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or up to 100%.Accordingly, the term “reduced” encompasses both a partial reduction anda complete reduction of a disease state.

As used herein, the term “reduced” can also refer to a decrease in thecell surface expression of a polypeptide when compared to an appropriatecontrol cell. In the present context, a reduction is distinct fromknockout of polypeptide expression, wherein expression is reduced by100%. Rather, in the present invention, a reduction indicates thatexpression is decreased but not completely eliminated. Such as areduction can be, for example, a reduction in cell surface beta-2microglobulin, MHC class I molecule, or CD52 expression when compared toa control cell which has not been genetically-modified to reduce beta-2microglobulin, MHC class I molecules, or CD52, respectively. A reductionin expression can be between about 10% and about 99% or any number orrange therein. For example, a reduction can be between about 10% and95%, about 50% and about 95%, about 75% and about 95%, or about 90% andabout 95%, when compared to a control cell. A reduction can also be byabout 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% whencompared to a control cell.

As used herein, the term “MHC class I molecule” refers to a majorhistocompatibility complex (MHC) found on the cell surface whichdisplays peptide fragments of non-self proteins. MHC class I moleculesconsist of two polypeptide chains. The alpha chain consists of 3polypeptides referred to as the alpha-1 (□1), alpha-2 (□2), and alpha-3(□3) domains. The alpha chain is linked non-covalently via the □3 domainto a beta chain which consists of beta-2 microglobulin (B2M). The alphachain is polymorphic and is encoded by the HLA gene (i.e., HLA-A, HLA-B,and HLA-C), whereas beta-2 microglobulin is not polymorphic and itencoded by the B2M gene.

As used herein, the term “beta-2 microglobulin” refers to the beta chaincomponent of MHC class I molecules. Human beta-2 microglobulin isencoded by the B2M gene (e.g., NCBI Gene ID 567). Expression of beta-2microglobulin is necessary for assembly and function of MHC class Imolecules on the cell surface.

As used herein, the term “CD52” refers to the polypeptide encoded by thehuman CD52 gene (e.g., NCBI gene ID 1043) which is also referred to ascluster of differentiation 52.

As used herein, the term “anti-tumor activity” or “anti-tumor effect”refers to a biological effect which can be manifested by a decrease intumor volume, a decrease in the number of tumor cells, a decrease in thenumber of metastases, an increase in life expectancy, or amelioration ofvarious physiological symptoms associated with the cancerous condition.An “anti-tumor effect” can also be manifested by the ability of thegenetically-modified cells of the present disclosure in prevention ofthe occurrence of tumor in the first place.

The term “effective amount” or “therapeutically effective amount” refersto an amount sufficient to effect beneficial or desirable biologicaland/or clinical results. The therapeutically effective amount will varydepending on the therapeutic (e.g., genetically-modified cell, CAR Tcell, etc.) formulation or composition, the disease and its severity,and the age, weight, physical condition and responsiveness of thesubject to be treated. In specific embodiments, an effective amount of acell comprising a co-stimulatory domain disclosed herein orpharmaceutical compositions disclosed herein reduces at least onesymptom or the progression of a disease.

As used herein, the term “treat” or “treatment” means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

As used herein, the term “cancer” should be understood to encompass anyneoplastic disease (whether invasive or metastatic) which ischaracterized by abnormal and uncontrolled cell division causingmalignant growth or tumor.

As used herein, the term “carcinoma” refers to a malignant growth madeup of epithelial cells.

As used herein, the term “leukemia” refers to malignancies of thehematopoietic organs/systems and is generally characterized by anabnormal proliferation and development of leukocytes and theirprecursors in the blood and bone marrow.

As used herein, the term “sarcoma” refers to a tumor which is made up ofa substance like the embryonic connective tissue and is generallycomposed of closely packed cells embedded in a fibrillary,heterogeneous, or homogeneous substance.

As used herein, the term “melanoma” refers to a tumor arising from themelanocytic system of the skin and other organs.

As used herein, the term “lymphoma” refers to a group of blood celltumors that develop from lymphocytes.

As used herein, the term “blastoma” refers to a type of cancer that iscaused by malignancies in precursor cells or blasts (immature orembryonic tissue).

As used herein, “transfected” or “transformed” or “transduced” or“nucleofected” refers to a process by which exogenous nucleic acid istransferred or introduced into the host cell. A “transfected” or“transformed” or “transduced” cell is one which has been transfected,transformed or transduced with exogenous nucleic acid. The cell includesthe primary subject cell and its progeny.

As used herein, a “human T cell” or “T cell” refers to a T cell isolatedfrom a human donor. Human T cells, and cells derived therefrom, includeisolated T cells that have not been passaged in culture, T cells thathave been passaged and maintained under cell culture conditions withoutimmortalization, and T cells that have been immortalized and can bemaintained under cell culture conditions indefinitely.

As used herein, a “human natural killer cell” or “human NK cell” or“natural killer cell” or “NK cell” refers to a type of cytotoxiclymphocyte critical to the innate immune system. The role NK cells playis analogous to that of cytotoxic T-cells in the vertebrate adaptiveimmune response. NK cells provide rapid responses to virally infectedcells and respond to tumor formation, acting at around 3 days afterinfection.

As used herein, a “control” or “control cell” refers to a cell thatprovides a reference point for measuring changes in genotype orphenotype of a genetically-modified cell. A control cell may comprise,for example: (a) a wild-type cell, i.e., of the same genotype as thestarting material for the genetic alteration which resulted in thegenetically-modified cell; (b) a cell of the same genotype as thegenetically-modified cell but which has been transformed with a nullconstruct (i.e., with a construct which has no known effect on the traitof interest); or, (c) a cell genetically identical to thegenetically-modified cell but which is not exposed to conditions,stimuli, or further genetic modifications that would induce expressionof altered genotype or phenotype. In particular embodiments, a controlcell is otherwise identical to a genetically-modified cell but has notbeen genetically-modified to reduce cell surface expression of aparticular polypeptide (e.g., beta-2 microglobulin, MHC class Imolecules, CD52).

As used herein with respect to both amino acid sequences and nucleicacid sequences, the terms “percent identity,” “sequence identity,”“percentage similarity,” “sequence similarity,” and the like, refer to ameasure of the degree of similarity of two sequences based upon analignment of the sequences which maximizes similarity between alignedamino acid residues or nucleotides, and which is a function of thenumber of identical or similar residues or nucleotides, the number oftotal residues or nucleotides, and the presence and length of gaps inthe sequence alignment. A variety of algorithms and computer programsare available for determining sequence similarity using standardparameters. As used herein, sequence similarity is measured using theBLASTp program for amino acid sequences and the BLASTn program fornucleic acid sequences, both of which are available through the NationalCenter for Biotechnology Information (www.ncbi.nlm.nih.gov), and aredescribed in, for example, Altschul et al. (1990), J. Mol. Biol.215:403-410; Gish and States (1993), Nature Genet. 3:266-272; Madden etal. (1996), Meth. Enzymol.266:131-141; Altschul et al. (1997), NucleicAcids Res. 25:33 89-3402); Zhang et al. (2000), J. Comput. Biol.7(1-2):203-14. As used herein, percent similarity of two amino acidsequences is the score based upon the following parameters for theBLASTp algorithm: word size=3; gap opening penalty=−11; gap extensionpenalty=−1; and scoring matrix=BLOSUM62. As used herein, percentsimilarity of two nucleic acid sequences is the score based upon thefollowing parameters for the BLASTn algorithm: word size=11; gap openingpenalty=−5; gap extension penalty=−2; match reward=1; and mismatchpenalty=−3.

As used herein with respect to modifications of two proteins or aminoacid sequences, the term “corresponding to” is used to indicate that aspecified modification in the first protein is a substitution of thesame amino acid residue as in the modification in the second protein,and that the amino acid position of the modification in the firstproteins corresponds to or aligns with the amino acid position of themodification in the second protein when the two proteins are subjectedto standard sequence alignments (e.g., using the BLASTp program). Thus,the modification of residue “X” to amino acid “A” in the first proteinwill correspond to the modification of residue “Y” to amino acid “A” inthe second protein if residues X and Y correspond to each other in asequence alignment, and despite the fact that X and Y may be differentnumbers.

As used herein, the recitation of a numerical range for a variable isintended to convey that the present disclosure may be practiced with thevariable equal to any of the values within that range. Thus, for avariable which is inherently discrete, the variable can be equal to anyinteger value within the numerical range, including the end-points ofthe range. Similarly, for a variable which is inherently continuous, thevariable can be equal to any real value within the numerical range,including the end-points of the range. As an example, and withoutlimitation, a variable which is described as having values between 0 and2 can take the values 0, 1 or 2 if the variable is inherently discrete,and can take the values 0.0, 0.1, 0.01, 0.001, or any other real valuesand ≥0 and ≤2 if the variable is inherently continuous.

2.1 Principle of the Invention

The present disclosure is based, in part, on the observation thatknockdown of cell surface beta-2 microglobulin, and consequently MHCclass I molecules, can reduce allogenicity of genetically-modifiedcells, such as CAR T cells. Importantly, the inventors have discoveredthat an incomplete knockdown of beta-2 microglobulin and MEW class Imolecules (i.e., to a low percentage of wild-type expression, but notcomplete knockout) not only reduces allogenicity of genetically-modifiedcells, but also serves to dramatically reduce killing by NK cells, whichcan recognize cells that are B2M-negative as non-self and induce acytotoxic action.

The present invention is also based, in part, on the inventors'discovery that a population of CAR-positive T cells can be enriched byan advantageous negative-selection method when the CAR-encodingconstruct includes a coding sequence for an RNA interfering moleculeagainst CD52. In this manner, a population of CAR T cells can becontacted with beads conjugated to an anti-CD52 antibody in order tocapture CD52-positive cells. Separation of the beads, and thus theCD52-positive cells, results in an enriched population of CAR-positivecells having reduced cell surface expression of CD52.

Accordingly, a nucleic acid molecule is provided comprising a firstexpression cassette which encodes an engineered antigen receptor, suchas a chimeric antigen receptor, and a second expression cassette whichencodes an inhibitory nucleic acid molecule, such as an RNA interferingmolecule. Further, the nucleic acid molecule is flanked by 5′ and 3′homology arms to promote targeted insertion of the nucleic acid into thegenome of a cell at a double-strand break, such as a cleavage siteproduced by an engineered nuclease. In certain embodiments of theinvention, the inhibitory nucleic acid molecule can be against humanbeta-2 microglobulin, a component of the MEW class I molecule, or CD52.

Further disclosed herein are recombinant DNA constructs and viralvectors comprising the nucleic acid molecule, genetically-modified cellscomprising the nucleic acid molecule, and pharmaceutical compositionscomprising such cells. Also disclosed are genetically-modified cellsexpressing an engineered antigen receptor (e.g., a CAR or exogenous TCR)which have reduced cell-surface expression of beta-2 microglobulin, MEWclass I molecules, or CD52, and may or may not express the particularnucleic acid molecule of the invention.

In some embodiments, administration of genetically-modified cells of theinvention reduces the symptoms or severity of diseases, such cancers,which can be targeted by genetically-modified cells of the presentdisclosure.

Also disclosed herein are methods of immunotherapy for treating cancerin a subject in need thereof comprising administering to the subject apharmaceutical composition comprising a genetically-modified celldisclosed herein and a pharmaceutically acceptable carrier. In suchmethods, wherein a CAR is expressed and cell surface beta-2microglobulin and/or MHC class I molecules is reduced, incompleteknockout leads to a reduction in both allogenicity of the cells andkilling of the cells by NK cells.

Further disclosed are methods for producing an enriched population ofgenetically-modified cells, wherein a CAR is expressed and cell surfaceCD52 is reduced by RNA interference, allowing for negative selection ofCAR-positive cells having reduced CD52 expression.

2.2 Nucleic Acid Molecules

In certain embodiments, the invention provides a nucleic acid moleculecomprising: (a) a first expression cassette comprising a nucleic acidsequence encoding an engineered antigen receptor; (b) a secondexpression cassette comprising a nucleic acid sequence encoding aninhibitory nucleic acid molecule; (c) a 5′ homology arm; and (d) a 3′homology arm. The 5′ homology arm and the 3′ homology arm can beengineered at any suitable length to have homology to chromosomalregions flanking a nuclease recognition sequence in a gene of interest,which can be any desired gene in a target cell in which a suitablerecognition sequence is present.

The nucleic acid molecule of the invention can have any number oforientations. Some non-limiting examples illustrated in FIG. 1. Inparticular embodiments, the first and second expression cassettes can bein the same orientation. This orientation can be either 5′ to 3′relative to the homology arms or, alternatively, 3′ to 5′. In eithercase, the first expression cassette may be 5′ to the second cassette, orthe second cassette may be 5′ to the first cassette. In otherembodiments, the first and second expression cassettes can be indifferent orientations in the nucleic acid molecule. For example, thefirst expression cassette may be oriented 5′ to 3′, whereas the secondexpression cassette may be oriented 3′ to 5′. Alternatively, the firstexpression cassette may be oriented 3′ to 5′ and the second expressioncassette may be oriented 5′ to 3′.

In embodiments wherein the expression cassettes are in oppositeorientations, they may be oriented in a “tail-to-tail” configuration,such that the first expression cassette is oriented 3′ to 5′ and ispositioned 5′ to the second expression cassette, which is oriented 5′ to3′. In a similar tail-to-tail embodiment, the second expression cassetteis oriented 3′ to 5′ and is positioned 5′ to the first expressioncassette, which is oriented 5′ to 3′.

In other embodiments wherein the expression cassettes are in oppositeorientations, they may be oriented in a “head-to-head” configuration,such that the first expression cassette is oriented 5′ to 3′ and ispositioned 5′ to the second expression cassette, which is oriented 3′ to5′. In a similar head-to-head embodiment, the second expression cassetteis oriented 5′ to 3′ and is positioned 5′ to the first expressioncassette, which is oriented 3′ to 5′.

In some embodiments, the nucleic acid molecule can comprise multiplecopies of the second expression cassette. The copies of the secondexpression cassette can be identical or vary from one another. In somecases, the copies can include a promoter, a coding sequence for theinhibitory nucleic acid molecule, and a sequence, such as a (cPPT/CTS)sequence, to terminate translation of the inhibitory nucleic acidmolecule. The copies of the second expression cassette can be in tandemto one another in the nucleic acid molecule, and can be in the sameorientation, or in opposite orientations. Alternatively, the copies maynot be in tandem, and can be in the same orientation, or in oppositeorientations.

The expression cassettes of the nucleic acid molecule can includevarious promoters which drive expression of the engineered antigenreceptor and/or the inhibitory nucleic acid molecule. One example of asuitable promoter is the immediate early cytomegalovirus (CMV) promotersequence. This promoter sequence is a strong constitutive promotersequence capable of driving high levels of expression of anypolynucleotide sequence operatively linked thereto. Another example of asuitable promoter is Elongation Growth Factor-1α (EF-1α). However, otherconstitutive promoter sequences may also be used, including, but notlimited to the simian virus 40 (SV40) early promoter, mouse mammarytumor virus (MMTV), human immunodeficiency virus (HIV) long terminalrepeat (LTR) promoter, MoMuLV promoter, an avian leukemia viruspromoter, an Epstein-Barr virus immediate early promoter, a Rous sarcomavirus promoter, as well as human gene promoters such as, but not limitedto, the actin promoter, the myosin promoter, the hemoglobin promoter,and the creatine kinase promoter. Further, the present disclosure shouldnot be limited to the use of constitutive promoters. Inducible promotersare also contemplated as part of the present disclosure. The use of aninducible promoter provides a molecular switch capable of turning onexpression of the polynucleotide sequence which it is operatively linkedwhen such expression is desired, or turning off the expression whenexpression is not desired. Examples of inducible promoters include, butare not limited to a metallothionine promoter, a glucocorticoidpromoter, a progesterone promoter, and a tetracycline promoter.

Synthetic promoters are also contemplated as part of the presentdisclosure. For example, in particular embodiments, the promoter drivingexpression of the engineered antigen receptor is a JeT promoter (see,WO/2002/012514).

In some embodiments, the promoters are selected based on the desiredoutcome. It is recognized that different applications can be enhanced bythe use of different promoters in the expression cassettes to modulatethe timing, location and/or level of expression of the polynucleotidesdisclosed herein. Such expression constructs may also contain, ifdesired, a promoter regulatory region (e.g., one conferring inducible,constitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific/selective expression), a transcription initiation startsite, a ribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

Promoters particularly useful for driving expression of an RNAinterference molecule are well known in the art and can include, withoutlimitation, pol III promoters, such as U6 or H1.

The 5′ and 3′ homology arms of the nucleic acid molecule have sequencehomology to corresponding sequences 5′ upstream and 3′ downstream of thenuclease recognition sequence in the genome. The homology arms promoteinsertion of the nucleic acid molecule into the cleavage site generatedby the nuclease. In general, homology arms can have a length of at least50 base pairs, preferably at least 100 base pairs, and up to 2000 basepairs or more, and can have at least 90%, preferably at least 95%, ormore, sequence homology to their corresponding sequences in the genome.

In order to assess the expression of an engineered antigen receptor(e.g. a CAR or exogenous T cell receptor) in a genetically-modifiedcell, the nucleic acid molecule of the invention can optionally comprisean epitope which can be used to detect the presence of the encoded cellsurface protein. In some examples described herein, a CAR codingsequence may include a QBend10 epitope which allows for detection usingan anti-CD34 antibody (see, W02013/153391).

In other examples, an expression cassette can also contain either aselectable marker gene or a reporter gene, or both, to facilitateidentification and selection of expressing cells from the population ofcells sought to be transfected or infected through viral vectors. Inother aspects, the selectable marker may be carried on a separate pieceof DNA and used in a co-transfection procedure. Both selectable markersand reporter genes may be flanked with appropriate regulatory sequencesto enable expression in the host cells. Useful selectable markersinclude, for example, antibiotic-resistance genes and fluorescent markergenes.

Expression may also be assessed by determining protein expression of thepolypeptide targeted by the inhibitory nucleic acid sequence. Forexample, expression of beta-2 microglobulin and CD52 can be detected onthe cell surface by a number of techniques known in the art. Expressioncan also be determined by positive or negative selection procedureswhich purify particular populations of cells expressing, or lackingexpression, of the cell surface polypeptides.

Also provided herein are vectors comprising the nucleic acid moleculesof the present disclosure. In some embodiments, the nucleic acidmolecule is cloned into a vector including, but not limited to aplasmid, a phagemid, a phage derivative, an animal virus, or a cosmid.Vectors of particular interest include expression vectors, replicationvectors, probe generation vectors, and sequencing vectors.

In other embodiments, nucleic acid molecules of the invention areprovided on viral vectors, such as retroviral vectors, lentiviralvectors, adenoviral vectors, and adeno-associated viral (AAV) vectors.Viral vector technology is well known in the art and is described, forexample, in Sambrook et al. (2001, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, New York), and in other virologyand molecular biology manuals. Viruses, which are useful as vectorsinclude, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers, (e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193). Where the nucleicacid of the invention is provided in a viral vector that promotes randomintegration into the genome, and does not require the presence of 5′ and3′ homology arms for homologous recombination, the nucleic acid of theinvention can be provided without 5′ and 3′ homology arms.

2.3 Chimeric Antigen Receptors (CARs)

Provided herein are genetically-modified cells expressing an engineeredantigen receptor. In some embodiments, the engineered antigen receptoris a chimeric antigen receptor (CAR). Generally, a CAR of the presentdisclosure will comprise at least an extracellular domain and anintracellular domain. In some embodiments, the extracellular domaincomprises a target-specific binding element otherwise referred to as aligand-binding domain or moiety. In some embodiments, the intracellulardomain, or cytoplasmic domain, comprises at least one co-stimulatorydomain and one or more signaling domains. In other embodiments, the CARmay only comprise a signaling domain, such as CD3□, and the cell maycomprise one or more co-stimulatory domains on another constructexpressed in the cell.

In some embodiments, a CAR comprises an extracellular, target-specificbinding element otherwise referred to as a ligand-binding domain ormoiety. The choice of ligand-binding domain depends upon the type andnumber of ligands that define the surface of a target cell. For example,the ligand-binding domain may be chosen to recognize a ligand that actsas a cell surface marker on target cells associated with a particulardisease state. Thus, examples of cell surface markers that may act asligands for the ligand-binding domain in the CAR of the presentdisclosure can include those associated with viral, bacterial andparasitic infections, autoimmune disease, and cancer cells. In someembodiments, the CAR of the present disclosure is engineered to target atumor antigen of interest by way of engineering a desired ligand-bindingmoiety that specifically binds to an antigen on a tumor cell. In thecontext of the present disclosure, “tumor antigen” refers to antigensthat are common to specific hyperproliferative disorders such as cancer.

In some embodiments, the extracellular ligand-binding domain of the CARis specific for any antigen or epitope of interest, particularly anytumor antigen or epitope of interest. As non-limiting examples, in someembodiments the antigen of the target is a tumor-associated surfaceantigen, such as ErbB2 (HER2/neu), carcinoembryonic antigen (CEA),epithelial cell adhesion molecule (EpCAM), epidermal growth factorreceptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD22, CD30,CD40, CLL-1, disialoganglioside GD2, ductal-epithelial mucine, gp36,TAG-72, glycosphingolipids, glioma-associated antigen, B-human chorionicgonadotropin, alphafetoprotein (AFP), lectin-reactive AFP,thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase,RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF,prostase, prostase specific antigen (PSA), PAP, NY-ESO-1, LAGA-la, p53,prostein, PSMA, surviving and telomerase, prostate-carcinoma tumorantigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, insulingrowth factor (IGFI)-1, IGF-II, IGFI receptor, mesothelin, a majorhistocompatibility complex (MHC) molecule presenting a tumor-specificpeptide epitope, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigens, theextra domain A (EDA) and extra domain B (EDB) of fibronectin and the Aldomain of tenascin-C (TnC Al) and fibroblast associated protein (fap); alineage-specific or tissue specific antigen such as CD3, CD4, CD8, CD24,CD25, CD33, CD34, CD38, CD123, CD133, CD138, CTLA-4, B7- 1 (CD80), B7-2(CD86), endoglin, a major histocompatibility complex (MHC) molecule,BCMA (CD269, TNFRSF 17), CS1, or a virus-specific surface antigen suchas an HIV-specific antigen (such as HIV gp120); an EBV-specific antigen,a CMV-specific antigen, a HPV-specific antigen such as the E6 or E7oncoproteins, a Lasse Virus-specific antigen, an InfluenzaVirus-specific antigen, as well as any derivate or variant of thesesurface markers. In a particular embodiment of the present disclosure,the ligand-binding domain is specific for CD19.

In some embodiments, the extracellular domain of a chimeric antigenreceptor further comprises an autoantigen (see, Payne et al. (2016)Science, Vol. 353 (6295): 179-184), which can be recognized byautoantigen-specific B cell receptors on B lymphocytes, thus directing Tcells to specifically target and kill autoreactive B lymphocytes inantibody-mediated autoimmune diseases. Such CARs can be referred to aschimeric autoantibody receptors (CAARs), and the incorporation of one ormore co-stimulatory domains described herein into such CAARs isencompassed by the present disclosure.

In some embodiments, the extracellular domain of a chimeric antigenreceptor can comprise a naturally-occurring ligand for an antigen ofinterest, or a fragment of a naturally-occurring ligand which retainsthe ability to bind the antigen of interest.

In some embodiments, a CAR comprises a transmembrane domain which linksthe extracellular ligand-binding domain or autoantigen with theintracellular signaling and co-stimulatory domains via a hinge or spacersequence. The transmembrane domain can be derived from anymembrane-bound or transmembrane protein. For example, the transmembranepolypeptide can be a subunit of the T-cell receptor (i.e., an α, β, γ orξ, polypeptide constituting CD3 complex), IL2 receptor p55 (a chain),p75 (β chain) or γ chain, subunit chain of Fc receptors (e.g., Fcyreceptor III) or CD proteins such as the CD8 alpha chain. Alternativelythe transmembrane domain can be synthetic and can comprise predominantlyhydrophobic residues such as leucine and valine. In particular examples,the transmembrane domain is a CD8□ transmembrane polypeptide.

The hinge region refers to any oligo- or polypeptide that functions tolink the transmembrane domain to the extracellular ligand-bindingdomain. For example, a hinge region may comprise up to 300 amino acids,preferably 10 to 100 amino acids and most preferably 25 to 50 aminoacids. Hinge regions may be derived from all or part of naturallyoccurring molecules, such as from all or part of the extracellularregion of CD8, CD4 or CD28, or from all or part of an antibody constantregion. Alternatively, the hinge region may be a synthetic sequence thatcorresponds to a naturally occurring hinge sequence, or may be anentirely synthetic hinge sequence. In particular examples, a hingedomain can comprise a part of a human CD8 alpha chain, FcyRIIIa receptoror IgGl.

The intracellular signaling domain of a CAR of the present disclosure isresponsible for activation of at least one of the normal effectorfunctions of the cell in which the CAR has been placed and/or activationof proliferative and cell survival pathways. The term “effectorfunction” refers to a specialized function of a cell. Effector functionof a T cell, for example, may be cytolytic activity or helper activityincluding the secretion of cytokines. An intracellular signaling domain,such as CD3 , can provide an activation signal to the cell in responseto binding of the extracellular domain. As discussed, the activationsignal can induce an effector function of the cell such as, for example,cytolytic activity or cytokine secretion.

The intracellular domain of the CAR can include one or moreintracellular co-stimulatory domains which transmit a co-stimulatorysignal to promote cell proliferation, cell survival, and/or cytokinesecretion after binding of the extracellular domain. Such intracellularco-stimulatory domains include those known in the art such as, withoutlimitation, CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1,ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, B7-H3 and a ligand that specifically binds with CD83, N1, or N6.

The CAR can be specific for any type of cancer cell. Such cancers caninclude, without limitation, carcinoma, lymphoma, sarcoma, blastomas,leukemia, cancers of B cell origin, breast cancer, gastric cancer,neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer,colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma,leukemia, and Hodgkin's lymphoma. In certain embodiments, cancers of Bcell origin include, without limitation, B lineage acute lymphoblasticleukemia, B cell chronic lymphocytic leukemia, B cell non-Hodgkin'slymphoma, and multiple myeloma.

2.4 Methods for Producing Recombinant Viral Vectors

In some embodiments, the present disclosure provides recombinant AAVvectors for use in the methods of the present disclosure. RecombinantAAV vectors are typically produced in mammalian cell lines such asHEK-293. Because the viral cap and rep genes are removed from the vectorto prevent its self-replication to make room for the therapeutic gene(s)to be delivered (e.g. the endonuclease gene), it is necessary to providethese in trans in the packaging cell line. In addition, it is necessaryto provide the “helper” (e.g. adenoviral) components necessary tosupport replication (Cots D, Bosch A, Chillon M (2013) Curr. Gene Ther.13(5): 370-81). Frequently, recombinant AAV vectors are produced using atriple-transfection in which a cell line is transfected with a firstplasmid encoding the “helper” components, a second plasmid comprisingthe cap and rep genes, and a third plasmid comprising the viral ITRscontaining the intervening DNA sequence to be packaged into the virus.Viral particles comprising a genome (ITRs and intervening gene(s) ofinterest) encased in a capsid are then isolated from cells byfreeze-thaw cycles, sonication, detergent, or other means known in theart. Particles are then purified using cesium-chloride density gradientcentrifugation or affinity chromatography and subsequently delivered tothe gene(s) of interest to cells, tissues, or an organism such as ahuman patient. Accordingly, methods are provided herein for producingrecombinant AAV vectors comprising the nucleic acid molecules of theinvention described herein.

In some embodiments, genetic transfer is accomplished via lentiviralvectors. Lentiviruses, in contrast to other retroviruses, in somecontexts may be used for transducing certain non-dividing cells.Non-limiting examples of lentiviral vectors include those derived from alentivirus, such as Human Immunodeficiency Virus 1 (HIV-1), HIV-2, anSimian Immunodeficiency Virus (SIV), Human T-lymphotropic virus 1(HTLV-1), HTLV-2 or equine infection anemia virus (E1AV). For example,lentiviral vectors have been generated by multiply attenuating the HIVvirulence genes, for example, the genes env, vif, vpr, vpu and nef aredeleted, making the vector safer for therapeutic purposes. Lentiviralvectors are known in the art, see Naldini et al., (1996 and 1998);Zufferey et al., (1997); Dull et al., 1998, U.S. Pat. Nos. 6,013,516;and 5,994,136). In some embodiments, these viral vectors areplasmid-based or virus-based, and are configured to carry the essentialsequences for incorporating foreign nucleic acid, for selection, and fortransfer of the nucleic acid into a host cell. Known lentiviruses can bereadily obtained from depositories or collections such as the AmericanType Culture Collection (“ATCC”; 10801 University Blvd., Manassas, Va.20110-2209), or isolated from known sources using commonly availabletechniques.

In specific embodiments, lentiviral vectors are prepared using a firstplasmid encoding the gag, pol, tat, and rev genes cloned from humanimmunodeficiency virus (HIV) and a second plasmid encoding the envelopeprotein from vesicular stomatitis virus (VSV-G) used to pseudotype viralparticles. A transfer vector, such as the pCDH-EF1-MCS vector, can beused with a suitable promoter. All three plasmids can then betransfected into lentivirus cells, such as the Lenti-X-293T cells, andlentivirus can then be harvested, concentrated and screened after asuitable incubation time. Accordingly, methods are provided herein forproducing recombinant lentiviral vectors comprising the nucleic acidmolecule of the invention described herein.

2.5 Genetically-Modified Cells

Provided herein are cells genetically-modified to comprise the nucleicacid molecule of the invention described herein. Further provided aregenetically-modified cells (e.g., human T cells expressing a CAR orexogenous TCR) with reduced cell-surface expression of beta-2microglobulin, MHC class I molecules, and or CD52, which do notnecessarily comprise the particular nucleic acid molecule of theinvention.

In different variations of the present disclosure, a nucleic acidmolecule of the invention is present within the genome of thegenetically-modified cell or, alternatively, is not integrated into thegenome of the cell. In particular embodiments, the nucleic acid moleculeof the invention is inserted into the genome of a cell by targetedinsertion at a cleavage site produced by a double-strand break, such asthat produced by an engineered nuclease. The presence of 5′ and 3′homology arms flanking the first and second expression cassettes of thenucleic acid molecule promote homologous recombination of the nucleicacid molecule into the cleavage site, resulting in targeted insertion.

In some embodiments where the nucleic acid molecule is not integratedinto the genome, the nucleic acid molecule can be present in thegenetically-modified cell in a recombinant DNA construct, in an mRNA, ina viral genome, or other nucleic acid which is not integrated into thegenome of the cell.

In specific embodiments, the cells comprising the nucleic acid moleculeof the invention, and other genetically-modified cells of the invention,are eukaryotic cells. In particular embodiments, such cells are T cellsor NK cells, particularly human T cells or NK cells. In someembodiments, the cells are primary T cells or primary NK cells.

T cells and NK cells can be obtained from a number of sources, includingperipheral blood mononuclear cells, bone marrow, lymph node tissue, cordblood, thymus tissue, tissue from a site of infection, ascites, pleuraleffusion, spleen tissue, and tumors. In certain embodiments of thepresent disclosure, any number of T cell and NK cell lines available inthe art may be used. In some embodiments of the present disclosure, Tcells and NK cells are obtained from a unit of blood collected from asubject using any number of techniques known to the skilled artisan. Inone embodiment, cells from the circulating blood of an individual areobtained by apheresis.

Genetically-modified cells comprising the nucleic acid moleculedisclosed herein, and other genetically-modified cells of the invention,can exhibit a number of functional properties dependent upon whichpolypeptide is reduced in the cell and/or targeted by the inhibitorynucleic acid molecule. For example, in some genetically-modified cellsof the invention, beta-2 microglobulin is reduced, or the inhibitorynucleic acid molecule is against human beta-2 microglobulin, and cellsurface beta-2 microglobulin expression is reduced, to a smallpercentage of wild-type expression. Such genetically-modified cells canbe less susceptible to endogenous NK cell killing, have extendedpersistence time in a subject, exhibit enhanced expansion in a subject,and/or have reduced allogenicity than cells with wild-type levels of B2Mor cells which are completely B2M-negative. Reductions in beta-2microglobulin consequently result in a reduction in cell surfaceexpression of MHC class I molecules, because beta-2 microglobulin isnecessary for their assembly and function. Therefore, the sameproperties are also applicable to genetically-modified cells of theinvention which have reduced cell surface expression of MHC class Imolecules.

Susceptibility to NK cell killing can be determined by methods known inthe art such as those described further herein. Reductions in NK cellkilling can be by about 5%, 10%, 20%, 30%, 40%, 50% 55%, 60%, 65%, 70%,75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% 94%, 95%, 96, 97%,98%, 99%, or up to 100% when compared to a control cell.

The genetically-modified cells of the invention are capable of expansionin a subject following administration. Here, expansion is considered anincrease in cell number resulting from proliferation and division invivo. The degree of expansion depends, in part, on the subject'sresponse to the cells; for example, if the cells are identified asallogeneic and/or non-self, the subject's immune system may reduce theability of the cells to expand and further reduce persistence of thecells post-administration. Thus, in some examples, genetically-modifiedcells of the invention can exhibit an increase in expansion in a subjectthat is about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%150%, 200%, 250%, 200%, 350%, 400%, 450%, 500%, up to 1000%, or more,when compared to a control cell. Expansion in vivo can be determinedpost-administration by any method known in the art. Persistence time ofa genetically-modified cell in a subject can be considered, for example,as the amount of time post-administration of the cell that it can bedetected in the subject by any method known in the art. In someexamples, a genetically-modified cell of the invention will have anincrease in persistence time that is up to about 1 week, 2 weeks, 3,weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 2 months, 3 months, 4 months,5 months, 6 months, 1 year, or more, longer than a control cell.

Allogenicity can be determine by any method known in the art, such asthose methods described further herein. The genetically-modified cellsof the invention can exhibit a reduction in allogenicity of about 5%,10%, 20%, 30%, 40%, 50% 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93% 94%, 95%, 96, 97%, 98%, 99%, or up to 100%when compared to a control cell.

2.6 Methods for Producing Genetically-Modified Cells

The present disclosure provides methods for producinggenetically-modified cells comprising the nucleic acid molecule of theinvention described herein. In specific embodiments, methods areprovided for modifying the cell to comprise the nucleic acid molecule.In different aspects of the present disclosure, the nucleic acidmolecule is integrated into the genome of the cell or, alternatively, isnot integrated into the genome of the cell.

In some embodiments, the nucleic acid molecule is introduced into a cellusing any technology known in the art. In specific embodiments, vectorsor expression cassettes comprising the nucleic acid molecule disclosedherein is introduced into a cell using a viral vector. Such vectors areknown in the art and include lentiviral vectors, adenoviral vectors, andadeno-associated virus (AAV) vectors (reviewed in Vannucci, et al. (2013New Microbiol. 36:1-22). Recombinant AAV vectors useful in the presentdisclosure can have any serotype that allows for transduction of thevirus into the cell and insertion of the nuclease gene into the celland, in particular embodiments, into the cell genome. In particularembodiments, recombinant AAV vectors have a serotype of AAV2, AAV6, orAAV8. Recombinant AAV vectors can also be self-complementary such thatthey do not require second-strand DNA synthesis in the host cell(McCarty, et al. (2001) Gene Ther. 8:1248-54).

In some embodiments, nucleic acid molecules disclosed herein aredelivered into a cell in the form of DNA (e.g., circular or linearizedplasmid DNA or PCR products) and/or via a viral vector. In someembodiments, the nucleic acid molecule disclosed herein is coupledcovalently or non-covalently to a nanoparticle or encapsulated withinsuch a nanoparticle using methods known in the art (Sharma, et al.(2014) Biomed Res Int. 2014). A nanoparticle is a nanoscale deliverysystem whose length scale is <1 □m, preferably <100 nm. Suchnanoparticles may be designed using a core composed of metal, lipid,polymer, or biological macromolecule, and multiple copies of the nucleicacid molecules can be attached to or encapsulated with the nanoparticlecore. This increases the copy number of the DNA that is delivered toeach cell and, so, increases the intracellular expression to maximizethe likelihood that the encoded products will be expressed. The surfaceof such nanoparticles may be further modified with polymers or lipids(e.g., chitosan, cationic polymers, or cationic lipids) to form acore-shell nanoparticle whose surface confers additional functionalitiesto enhance cellular delivery and uptake of the payload (Jian et al.(2012) Biomaterials. 33(30): 7621-30). Nanoparticles may additionally beadvantageously coupled to targeting molecules to direct the nanoparticleto the appropriate cell type and/or increase the likelihood of cellularuptake. Examples of such targeting molecules include antibodies specificfor cell surface receptors and the natural ligands (or portions of thenatural ligands) for cell surface receptors.

In some embodiments, the nucleic acid molecule disclosed herein can beencapsulated within liposomes or complexed using cationic lipids (see,e.g., LIPOFECTAMINE, Life Technologies Corp., Carlsbad, Calif.; Zuris etal. (2015) Nat Biotechnol. 33: 73-80; Mishra et al. (2011) J Drug Deliv.2011:863734). The liposome and lipoplex formulations can protect thepayload from degradation, and facilitate cellular uptake and deliveryefficiency through fusion with and/or disruption of the cellularmembranes of the cells.

In some embodiments, the nucleic acid molecule disclosed herein can beencapsulated within polymeric scaffolds (e.g., PLGA) or complexed usingcationic polymers (e.g., PEI, PLL) (Tamboli et al. (2011) Ther Deliv.2(4): 523-536). In some embodiments, the nucleic acid molecule disclosedherein can be combined with amphiphilic molecules that self-assembleinto micelles (Tong et al. (2007) J Gene Med. 9(11): 956-66). Polymericmicelles may include a micellar shell formed with a hydrophilic polymer(e.g., polyethyleneglycol) that can prevent aggregation, mask chargeinteractions, and reduce nonspecific interactions outside of the cell.

In some embodiments, the nucleic acid molecule disclosed herein can beformulated as an emulsion for delivery to the cell. The term “emulsion”refers to, without limitation, any oil-in-water, water-in-oil,water-in-oil-in-water, or oil-in-water-in-oil dispersions or droplets,including lipid structures that can form as a result of hydrophobicforces that drive apolar residues (e.g., long hydrocarbon chains) awayfrom water and polar head groups toward water, when a water immisciblephase is mixed with an aqueous phase. These other lipid structuresinclude, but are not limited to, unilamellar, paucilamellar, andmultilamellar lipid vesicles, micelles, and lamellar phases. Emulsionsare composed of an aqueous phase and a lipophilic phase (typicallycontaining an oil and an organic solvent). Emulsions also frequentlycontain one or more surfactants. Nanoemulsion formulations are wellknown, e.g., as described in US Patent Application Nos. 2002/0045667 and2004/0043041, and U.S. Pat. Nos. 6,015,832, 6,506,803, 6,635,676, and6,559,189, each of which is incorporated herein by reference in itsentirety.

In some embodiments, the nucleic acid molecule disclosed herein can becovalently attached to, or non-covalently associated with,multifunctional polymer conjugates, DNA dendrimers, and polymericdendrimers (Mastorakos et al. (2015) Nanoscale. 7(9): 3845-56; Cheng etal. (2008) J Pharm Sci. 97(1): 123-43). The dendrimer generation cancontrol the payload capacity and size, and can provide a high payloadcapacity. Moreover, display of multiple surface groups can be leveragedto improve stability and reduce nonspecific interactions.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans. Physical methods for introducing a polynucleotide into a hostcell include calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York). A preferred method for the introduction of a polynucleotideinto a host cell is calcium phosphate transfection. Biological methodsfor introducing a polynucleotide of interest into a host cell includethe use of DNA and RNA vectors. Viral vectors, and especially retroviralvectors, have become the most widely used method for inserting genesinto mammalian, e.g., human cells. Other viral vectors can be derivedfrom lentivirus, poxviruses, herpes simplex virus I, adenoviruses andadeno-associated viruses, and the like. See, for example, U.S. Pat. Nos.5,350,674 and 5,585,362. Chemical means for introducing a polynucleotideinto a host cell include colloidal dispersion systems, such asmacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. An exemplary colloidal system for use as adelivery vehicle in vitro and in vivo is a liposome (e.g., an artificialmembrane vesicle).

2.7 Pharmaceutical Compositions

In some embodiments, the present disclosure provides a pharmaceuticalcomposition comprising a genetically-modified cell, or a population ofgenetically-modified cells, of the present disclosure and apharmaceutically-acceptable carrier. Such pharmaceutical compositionscan be prepared in accordance with known techniques. See, e.g.,Remington, The Science And Practice of Pharmacy (21st ed. 2005). In themanufacture of a pharmaceutical formulation according to the presentdisclosure, cells are typically admixed with a pharmaceuticallyacceptable carrier and the resulting composition is administered to asubject. The carrier must, of course, be acceptable in the sense ofbeing compatible with any other ingredients in the formulation and mustnot be deleterious to the subject. In some embodiments, pharmaceuticalcompositions of the present disclosure further comprises one or moreadditional agents useful in the treatment of a disease in the subject.In additional embodiments, where the genetically-modified cell is agenetically-modified human T cell or NK cell (or a cell derivedtherefrom), pharmaceutical compositions of the present disclosurefurther include biological molecules, such as cytokines (e.g., IL-2,IL-7, IL-15, and/or IL-21), which promote in vivo cell proliferation andengraftment. Pharmaceutical compositions comprising genetically-modifiedcells of the present disclosure can be administered in the samecomposition as an additional agent or biological molecule or,alternatively, can be co-administered in separate compositions.

In some embodiments, the pharmaceutical compositions of the presentdisclosure are useful for treating any disease state that can betargeted by T cell adoptive immunotherapy. In a particular embodiment,the pharmaceutical compositions of the present disclosure are useful asimmunotherapy in the treatment of cancer. Such cancers can include,without limitation, carcinoma, lymphoma, sarcoma, blastomas, leukemia,cancers of B-cell origin, breast cancer, gastric cancer, neuroblastoma,osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer,renal cell carcinoma, ovarian cancer, rhabdomyo sarcoma, leukemia, andHodgkin's lymphoma. In certain embodiments, cancers of B-cell origininclude, without limitation, B-lineage acute lymphoblastic leukemia,B-cell chronic lymphocytic leukemia, and B-cell non-Hodgkin's lymphoma.Non-limiting examples of cancer which may be treated with thepharmaceutical compositions and medicaments of the present disclosureare carcinomas, lymphomas, sarcomas, melanomas, blastomas, leukemias,and germ cell tumors, including but not limited to cancers of B-cellorigin, neuroblastoma, osteosarcoma, prostate cancer, renal cellcarcinoma, rhabdomyosarcoma, liver cancer, gastric cancer, bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, breastcancer, lung cancer, cutaneous or intraocular malignant melanoma, renalcancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer,rectal cancer, cancer of the anal region, stomach cancer, testicularcancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma ofthe endometrium, carcinoma of the cervix, carcinoma of the vagina,carcinoma of the vulva, non-Hodgkin's lymphoma, cancer of the esophagus,cancer of the small intestine, cancer of the endocrine system, cancer ofthe thyroid gland, cancer of the parathyroid gland, cancer of theadrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer ofthe penis, solid tumors of childhood, lymphocytic lymphoma, cancer ofthe bladder, cancer of the kidney or ureter, carcinoma of the renalpelvis, neoplasm of the central nervous system (CNS), primary CNSlymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma,pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cellcancer, environmentally induced cancers including those induced byasbestos, multiple myeloma, Hodgkin lymphoma, non-Hodgkin's lymphomas,acute myeloid lymphoma, chronic myelogenous leukemia, chronic lymphoidleukemia, immunoblastic large cell lymphoma, acute lymphoblasticleukemia, mycosis fungoides, anaplastic large cell lymphoma, and T-celllymphoma, and any combinations of said cancers. In certain embodiments,cancers of B-cell origin include, without limitation, B-lineage acutelymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-celllymphoma, diffuse large B cell lymphoma, pre-B ALL (pediatricindication), mantle cell lymphoma, follicular lymphoma, marginal zonelymphoma, Burkitt's lymphoma, and B-cell non-Hodgkin's lymphoma.

In some of these embodiments wherein cancer is treated with thepresently disclosed genetically-modified cells, the subject administeredthe genetically-modified cells is further administered an additionaltherapeutic, such as radiation, surgery, or a chemotherapeutic agent.

The invention further provides a population of genetically-modifiedcells comprising a plurality of genetically-modified cells describedherein. Such genetically-modified cells can comprise in their genome anucleic acid molecule encoding an engineered antigen receptor, such as achimeric antigen receptor or exogenous T cell receptor, and aninhibitory nucleic acid molecule, such as an RNA interference molecule.Such genetically-modified cells can also comprise in their genome anucleic acid molecule encoding an engineered antigen receptor, such as achimeric antigen receptor or exogenous T cell receptor, and have reducedcell surface expression of beta-2 microglobulin, MHC class I molecules,or CD52, without necessarily comprising the particular nucleic acidmolecule of the invention. Thus, in various embodiments of theinvention, a population of genetically-modified cells is providedwherein at least 10%, at least 15%, at least 20%, at least 25%, at least30%, at least 35%, at least 40%, at least 45%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or up to 100%, of cells in thepopulation are a genetically-modified cell described herein.

2.8 Methods of Administering Genetically-Modified Cells

Another aspect disclosed herein is the administration of thegenetically-modified cells of the present disclosure to a subject inneed thereof. In particular embodiments, the pharmaceutical compositionsdescribed herein are administered to a subject in need thereof. Forexample, an effective amount of a genetically-modified cell orpopulation of genetically-modified cells of the invention which expressa cell surface chimeric antigen receptor or exogenous T cell receptor,can be administered to a subject having a disease. In particularembodiments, the disease can be cancer, such as a cancer of B-cellorigin. Thus, the present disclosure also provides a method forproviding a T cell-mediated immune response to a target cell populationor tissue in a mammal, comprising the step of administering to themammal a CAR T cell, wherein the CAR comprises an extracellularligand-binding domain that specifically interacts with a predeterminedtarget, such as a tumor antigen, and an intracellular domain thatcomprises at least one signaling domain, such as CD3ξ, and optionallyone or more co-stimulatory signaling domains. The administered CAR Tcells are able to reduce the proliferation, reduce the number, or killtarget cells in the recipient. Unlike antibody therapies,genetically-modified cells of the present disclosure are able toreplicate and expand in vivo, resulting in long-term persistence thatcan lead to sustained control of a disease.

In examples wherein the inhibitory nucleic acid molecule is againsthuman beta-2 microglobulin or a component of the MHC class I molecule,or wherein beta-2 microglobulin or MHC class I molecules are otherwisereduced, expansion and/or persistence of such CAR T cells can beenhanced in the subject when compared to a CAR T cell having wild-typelevels of beta-2 microglobulin or MHC class I molecules, or no cellsurface beta-2 microglobulin or MHC class I expression. Further,allogenicity of the CAR T cell can be reduced when compared to a CAR Tcell having a wild-type level of cell surface expression of beta-2microglobulin and MHC class I molecules. These advantageouscharacteristics result from the incomplete reduction of cell surfacebeta-2 microglobulin (and consequently MHC class I molecules) to a smallpercentage of wild-type expression, which allows for reducedallogenicity but avoidance of NK cells which would otherwise target abeta-2 microglobulin-negative cell.

Examples of possible routes of administration include parenteral, (e.g.,intravenous (IV), intramuscular (IM), intradermal, subcutaneous (SC), orinfusion) administration. Moreover, the administration may be bycontinuous infusion or by single or multiple boluses. In specificembodiments, one or both of the agents is infused over a period of lessthan about 12 hours, 6 hours, 4 hours, 3 hours, 2 hours, or 1 hour. Instill other embodiments, the infusion occurs slowly at first and then isincreased over time.

In some embodiments, a genetically-modified eukaryotic cell orpopulation thereof of the present disclosure targets a tumor antigen forthe purposes of treating cancer. Such cancers can include, withoutlimitation, carcinoma, lymphoma, sarcoma, blastomas, leukemia, cancersof B cell origin, breast cancer, gastric cancer, neuroblastoma,osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer,renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, andHodgkin's lymphoma. In specific embodiments, cancers and disordersinclude but are not limited to pre-B ALL (pediatric indication), adultALL, mantle cell lymphoma, diffuse large B cell lymphoma, salvage postallogenic bone marrow transplantation, and the like. These cancers canbe treated using a combination of CARS that target, for example, CD19,CD20, CD22, and/or ROR1. In some non-limiting examples, agenetically-modified eukaryotic cell or population thereof of thepresent disclosure targets carcinomas, lymphomas, sarcomas, melanomas,blastomas, leukemias, and germ cell tumors, including but not limited tocancers of B-cell origin, neuroblastoma, osteosarcoma, prostate cancer,renal cell carcinoma, rhabdomyosarcoma, liver cancer, gastric cancer,bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck,breast cancer, lung cancer, cutaneous or intraocular malignant melanoma,renal cancer, uterine cancer, ovarian cancer, colorectal cancer, coloncancer, rectal cancer, cancer of the anal region, stomach cancer,testicular cancer, uterine cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, non-Hodgkin's lymphoma, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, solid tumors of childhood, lymphocyticlymphoma, cancer of the bladder, cancer of the kidney or ureter,carcinoma of the renal pelvis, neoplasm of the central nervous system(CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor,brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoidcancer, squamous cell cancer, environmentally induced cancers includingthose induced by asbestos, multiple myeloma, Hodgkin lymphoma,non-Hodgkin's lymphomas, acute myeloid lymphoma, chronic myelogenousleukemia, chronic lymphoid leukemia, immunoblastic large cell lymphoma,acute lymphoblastic leukemia, mycosis fungoides, anaplastic large celllymphoma, and T-cell lymphoma, and any combinations of said cancers. Incertain embodiments, cancers of B-cell origin include, withoutlimitation, B-lineage acute lymphoblastic leukemia, B-cell chroniclymphocytic leukemia, B-cell lymphoma, diffuse large B cell lymphoma,pre-B ALL (pediatric indication), mantle cell lymphoma, follicularlymphoma, marginal zone lymphoma, Burkitt's lymphoma, multiple myeloma,and B-cell non-Hodgkin's lymphoma.

When an “effective amount” or “therapeutic amount” is indicated, theprecise amount of the compositions of the present disclosure to beadministered can be determined by a physician with consideration ofindividual differences in age, weight, tumor size (if present), extentof infection or metastasis, and condition of the patient (subject). Insome embodiments, a pharmaceutical composition comprising thegenetically-modified cells described herein is administered at a dosageof 104 to 109 cells/kg body weight, including all integer values withinthose ranges. In further embodiments, the dosage is 105 to 106 cells/kgbody weight, including all integer values within those ranges. In someembodiments, cell compositions are administered multiple times at thesedosages. The cells can be administered by using infusion techniques thatare commonly known in immunotherapy (see, e.g., Rosenberg et al., NewEng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimefor a particular patient can readily be determined by one skilled in theart of medicine by monitoring the patient for signs of disease andadjusting the treatment accordingly.

In some embodiments, administration of genetically-modified cells of thepresent disclosure reduce at least one symptom of a target disease orcondition. For example, administration of genetically-modified cells ofthe present disclosure can reduce at least one symptom of a cancer, suchas cancers of B-cell origin. Symptoms of cancers, such as cancers ofB-cell origin, are well known in the art and can be determined by knowntechniques.

EXPERIMENTAL

This disclosure is further illustrated by the following examples, whichshould not be construed as limiting. Those skilled in the art willrecognize, or be able to ascertain, using no more than routineexperimentation, numerous equivalents to the specific substances andprocedures described herein. Such equivalents are intended to beencompassed in the scope of the claims that follow the examples below.

EXAMPLE 1 NK Cell Killing of B2M Knockout Primary Human T Cells 1.Methods and Materials

Primary human T cells were stimulated for 3 days using ImmunoCultanti-CD2/CD3/CD28 (StemCell Technologies) in the presence of IL-2(Gibco) in XVIVO-15 medium (Lonza) supplemented with 5% fetal bovineserum. RNA encoding B2M13-1433 479 nuclease was introduced into the Tcells using the 4D Nucleofector (Lonza). Cells were cultured in thepresence of IL-2 for 6 days before magnetic depletion of remaining B2M⁺cells using biotinylated anti-human B2M (BioLegend) and a BiotinSelection Kit (StemCell Technologies). NK cells were isolated from PBMCsamples of the same donor using a CD56 positive selection kit (StemCellTechnologies). Daudi cells were purchased from ATCC. Daudi cells arenaturally B2M⁻ and are reported to be highly sensitive to NK cytolysis.All target cells were labeled with 1 uM CellTrace Violet(LifeTechnologies) to distinguish them from effectors in mixed cultures.Isolated NK cells were mixed with either autologous B2M⁺ T cell targets(negative control for NK cytolysis), Daudi targets (positive control forNK cytolysis), or autologous B2M KO T cell targets (experimental sample)at effector:target ratios of 2:1, 1:1, 0.5:1, and 0:1. Killing wasassessed after 2h of co-culture. Killing by NK cells was measured bystaining with CaspACE—VAD-FMK (Promega).

2. Results

NK cells elicited only dim VAD-FMK signals in autologous B2M⁺ targets,indicating low levels of apoptosis induction by active caspases (FIGS.2A-2D Daudi cells (71-83%, FIGS. 2I-2L), indicating extensive caspasecascade activation. Similarly, B2M⁻ autologous T cells returned highVAD-FMK signals in response to NK encounter (19-47%, FIG. 2 E-H),indicative of caspase-mediated apoptosis induction. A graphical summaryof these results appears in FIG. 3.

3.Conclusions

Complete knockout of cell surface B2M using engineered meganucleasessensitizes primary human T cells to NK cell attack and killing.

EXAMPLE 2 Characterization of Candidate shRNAs Against B2M and Effect ofB2M Knockdown on NK Cell Killing of Primary Human T Cells 1. Materialsand Methods

Five Mission-shRNA lentiviral transfer plasmids encoding different B2Mtargeting sequences were purchased from Sigma-Aldrich. Second-generationlentiviral vectors were produced in-house using Lenti-X 293T cells(ClonTech) and a triple transfection method (Lipofectamine2000—Thermo-Fisher). T cells were prepared for lentiviral transductionby stimulating for 3 days with ImmunoCult anti-CD2/CD3/CD28 as inExample 1. Transduction was carried out in the presence of 5 uMpolybrene (Sigma-Aldritch) and transduced cells were selected withpuromycin (InVivoGen) beginning at 48 h post-transduction and concluding72h following drug addition. Selected cells were expanded for 5 days inIL-2 supplemented medium before a flow cytometric analysis of B2Msurface expression to determine the extent of knockdown. Culturesreceiving B2M shRNAs were used as targets in NK and CTL killing assays.The NK killing assays were carried out as described in Example 1, butthe K562 cell line was used as the positive control for NK cytolysis.For the CTL killing assay, CD8+ T cells from a donor unrelated to thedonor of the target cells were isolated and used as effectors. The NKkilling assay was carried out for 2 h and the CTL assay was carried outfor 6 h. For both assays, target cells were labeled with 1 uM CellTraceViolet (Life Technologies), and killing was measured usingCaspACE-VAD-FMK (Promega).

2. Results

Five shRNAs were screened in human T cells for interference with B2Mexpression. Two sequences did not reduce the mean fluorescence intensityof B2M in a cytometric analysis (not shown). Three shRNA sequences didreduce the 1MFI of B2M expression, with sequence 254 and 255 reducingMFI by approximately 50% and sequence 472 reducing the MFI byapproximately 95% (FIG. 4).

CTL and NK killing of targets exhibiting altered B2M expression was nextmeasured. NK cells, but not CTLs induced caspase activation (measured byVAD-FMK signal) in Class I deficient K562 cells (46% vs. 5%—FIGS. 5A and5B). Conversely, CTLs induced caspase activation (32%) in mismatchedB2M⁺ T cells while NK cells induced a signal in a lower frequency ofmismatched T cells (14%) (FIGS. 5C and 5D). In T cell targets exhibitinga 50% reduction in B2M antigen density, NK cells elicited caspaseactivity in 17% of targets while mismatched CTLs did so in 36% oftargets (FIGS. 6A and 6B). In T cell targets exhibiting a 95% knockdownof B2M levels, NK cells elicited caspase activation in 16% of targets,while mismatched CTLs did so in 20.8% of targets (FIGS. 5 C and D).

3. Conclusions

B2M expression can be effectively knocked down using shRNA delivered bya viral vector. Using caspase (VAD-FMK) activity to measure apoptosisinduction in target cells by NK cells or CTLs, it was determined thatB2M knockdown does not alter a target's susceptibility to NK cytolysis,as both B2M knockdown targets exhibited the same VAD-FMK frequency asun-manipulated targets, and less VAD-FMK signal than K562 targets. Inaddition, B2M knockdown confers some protection against CTL cytolysis,as the frequency of VAD-FMK+targets in the shRNA 472 group wasapproximately half the frequency observed in the positive control. Infact, there was a direct relationship between the degree of knockdownand the degree of protection against CTL activity from NK cells.

EXAMPLE 3 Production and Characterization of CAR T Cells Utilizing shRNAto Reduce Cell Surface Expression of B2M 1. Materials and Methods

A number of constructs were prepared comprising an anti-CD19 CAR codingsequence and an shRNA against B2M. These are illustrated in FIG. 7A-7Fand are provided in SEQ ID NOs: 18-23. CAR constructs 7007 and 7217 (SEQID NOs: 18 and 19) comprise the CAR coding sequence and the shRNA472sequence in the same 5′ to 3′ orientation. CAR constructs 7008 and 7218(SEQ ID NOs: 20 and 21) comprise the CAR coding sequence in the 3′ to 5′orientation, and shRNA472 sequence in the 5′ to 3′ orientation (i.e.,tail-to-tail). CAR constructs 7009 and 7219 (SEQ ID NOs: 22 and 23)comprise both the CAR coding sequence and the shRNA472 sequence in the3′ to 5′ orientation. The 5′ and 3′ homology arms flanking the CARcoding sequence and the shRNA472 sequence have homology to regionsupstream and downstream of the TRC 1-2 recognition sequence in the TRAClocus.

CAR T cells will be prepared using primary donor human T cellstransduced with recombinant AAV vectors comprising one of the CAR/shRNAconstructs above, with simultaneous nucleofection of mRNA encoding theTRC 1-2x.87EE to induce a double-strand break at the TRC 1-2 recognitionsequence and promote targeted insertion of the construct into the genomeof the T cells. Beta-2 microglobulin expression will be determined asdescribed above to determine which orientation of the first and secondexpression cassettes will result in the highest and/or the mostconsistent CAR expression, along with the most consistent level of B2Mknockdown on the cell surface.

CAR T cells produced with certain constructs will be evaluated in boththe allogenicity and NK cell killing assays previously described above.Further, CAR T cells produced using the disclosed constructs will beevaluated in various stress tests, in which the CAR T cells arerepeatedly exposed to antigen in order to determine changes in cellproliferation/expansion and cytotoxic potential. CAR T cells producedusing the disclosed constructs will also be utilized with in vivo tumormodels to determine their ability to clear tumor cells in an animal andto evaluate their ability to persist in vivo. It is expected, based onthe Examples described herein, that CAR T cells having a reduced butincomplete knockdown of cell surface beta-2 microglobulin will havegreater persistence and/or enhanced expansion in vivo when compared toCAR T cells which are completely B2M-negative and may be susceptible toNK cell killing.

In a particular study, CAR T cells were prepared that are TCR-negative,CAR-positive, and have reduced cell surface expression of B2M. CAR Tcells were prepared using donor templates that comprise apromoter-driven CAR coding sequence, a T2A element, and one or multiplepromoter-driven B2M shRNA cassettes. In this study, an apheresis samplewas drawn from a healthy, informed, and compensated donor, and the Tcells were enriched using the CD3 positive selection kit II in accordwith the manufacturer's instructions (Stem Cell Technologies). T cellswere activated using ImmunoCult T cell stimulator(anti-CD2/CD3/CD28—Stem Cell Technologies) in X-VIVO 15 medium (Lonza)supplemented with 5% fetal bovine serum and 10 ng/ml IL-2 (Gibco). After3 days of stimulation, cells were collected and samples of 1e6 cellswere electroporated with the following mixture of nucleic acid speciesusing the Lonza 4D NucleoFector.

-   -   1 μg mRNA encoding the TRC 1-2x.87EE meganuclease which produces        a double-strand break in Exon 1 of the T cell receptor alpha        constant region gene    -   1.5 μl of 100 mM siRNA specific for TMEM173 (STING)    -   1 μg of linearized plasmid DNA comprising a donor template In        this experiment, three different CAR constructs were analyzed        for their ability to knock down B2M surface expression. All        three constructs use homology to genomic regions flanking the        TRC 1-2x.87EE binding site (referred to as the TRC 1-2        recognition site) to direct targeted insertion into the T cell        receptor alpha constant region locus, and they all express a CAR        that comprises a CD34 epitope tag (for detection). Construct        7002 (SEQ ID NO: 11) does not encode an shRNA gene. Construct        7008 (SEQ ID NO: 20) encodes one copy of shRNA472. Construct        7029 (SEQ ID NO: 24) encodes two copies of this shRNA cassette.        Expression from each shRNA cassette is driven by a U6 promoter.

Cell cultures were maintained for 10 additional days in X-VIVO15 mediumsupplemented with 5% FBS and 30 ng/m1 of IL-2. On d4, 7, and 10post-nucleofection, the cultures were sampled and analyzed for surfaceexpression of CD3 (anti-CD3-BV711, BioLegend), CD34 (anti-CD34-PE,LifeTechnologies), and B2M (anti-B2M-APC, BioLegend). Flow cytometrydata were acquired on a Beckman-Coulter CytoFLEX-LX.

2. Results

B2M surface levels were measured in samples nucleofected with a controlCAR construct (7002) or with CAR constructs expressing one (7008) or two(7029) copies of B2M-specific shRNA (FIG. 12). When comparing theCD3-/CD34+ populations in 7002 (control) and 7008 (single shRNA)expressing cells, 7008 expressing cells were observed to displayslightly lower levels of surface B2M (FIG. 12A). Notably, cellsnucleofected with construct 7029 (two shRNA copies) displayedapproximately half of the amount of B2M displayed on the surface ofcontrol cells (7002) (FIG. 12B). This observation was specific to theCD3-/CD34+ population, but was not observed in the CD3-/CD34− population(FIG. 12C).

3. Conclusions

A pre-screened B2M-targeting shRNA can knock down B2M expression levelson the surface of cells into which the construct has been delivered (viatargeted insertion into the T cell receptor alpha constant regionlocus). Due to the high abundance of B2M transcripts, these data suggestthat a single shRNA copy can be sufficient for low levels of B2Mknockdown, whereas multiple copies of the shRNA cassette may be requiredto achieve more significant knockdown.

EXAMPLE 4 Production and Characterization of CAR T Cells Utilizing shRNAto Reduce Cell Surface Expression of B2M 1. Materials and Methods

A number of constructs were prepared comprising an anti-CD19 CAR codingsequence and an shRNA against B2M. These are illustrated in FIG. 7A-7Fand are provided in SEQ ID NOs: 18-23. CAR constructs 7007 and 7217 (SEQID NOs: 18 and 19) comprise the CAR coding sequence and the shRNA472sequence in the same 5′ to 3′ orientation. CAR constructs 7008 and 7218(SEQ ID NOs: 20 and 21) comprise the CAR coding sequence in the 3′ to 5′orientation, and shRNA472 sequence in the 5′ to 3′ orientation (i.e.,tail-to-tail). CAR constructs 7009 and 7219 (SEQ ID NOs: 22 and 23)comprise both the CAR coding sequence and the shRNA472 sequence in the3′ to 5′ orientation. CAR constructs 7056, 7059, and 7060 containmodified versions of the U6-shRNA gene cassette. A cloning site that waslocated between the U6 promoter and the hairpin sequence in constructs7007-7009, and in 7217-7219 was removed. Construct 7056 comprises theCAR coding sequence and the shRNA472 sequence in the 3′ to 5′orientation. Construct 7056 comprises the CAR coding sequence in 3′ to5′ orientation, and the shRNA472 sequence in the 5′ to 3′ orientation(tail-to-tail). Construct 7060 comprises the CAR coding sequence in 3′to 5′ orientation and two copies of the U6-shRNA472 sequence in 5′ to 3′orientation. The 5′ and 3′ homology arms flanking the CAR codingsequence and the shRNA472 sequence have homology to regions upstream anddownstream of the TRC 1-2 recognition sequence in the T cell receptoralpha constant locus.

In a particular study, CAR T cells were prepared that are TCR-negative,CAR-positive, and have reduced cell surface expression of B2M. CAR Tcells were prepared using donor templates that comprise apromoter-driven CAR coding sequence, and one or multiple promoter-drivenB2M shRNA cassettes. In this study, an apheresis sample was drawn from ahealthy, informed, and compensated donor, and the T cells were enrichedusing the CD3 positive selection kit II in accord with themanufacturer's instructions (Stem Cell Technologies). T cells wereactivated using ImmunoCult T cell stimulator (anti-CD2/CD3/CD28—StemCell Technologies) T cells were activated using ImmunoCult T cellsimulator (anti-CD2/CD3/CD28—Stem Cell Technologies( in X-VIVO 15 medium(Lonza) supplemented with 5% fetal bovine serum and 10 ng/ml IL-2(Gibco). After 3 days of stimulation, cells were collected and samplesof le6 cells were electroporated with the following mixture of nucleicacid species using the Lonza 4D NucleoFector.

-   -   1 μg mRNA encoding the TRC 1-2x.87EE meganuclease which produces        a double-strand break in Exon 1 of the T cell receptor alpha        constant region gene    -   1.5 μl of 100 mM siRNA specific for TMEM173 (STING)    -   1 μg of linearized plasmid DNA comprising a donor template        In this experiment, four different CAR constructs were analyzed        for their ability to knock down B2M surface expression. All four        constructs use homology to genomic regions flanking the TRC        1-2x.87EE recognition site to direct targeted insertion into the        T cell receptor alpha constant region locus, and they all        express a CAR that comprises a CD34 epitope tag (for detection).        Construct 7002 (SEQ ID NO: 11) does not encode an shRNA gene.        Construct 7056 (SEQ ID NO: 25) encodes one copy of shRNA472, and        both cassettes are in the 3′ to 5′ orientation (head-to-tail).        Construct 7059 (SEQ ID NO: 26) encodes one copy of this shRNA        cassette, with the CAR expression cassette in a 3′ to 5′        orientation, and the shRNA472 cassette in a 5′ to 3′ orientation        (tail-to-tail). Construct 7060 (SEQ ID NO: 27) is in the same        orientation as construct 7059 but encodes two copies of the        shRNA472 cassette (tail-to-tail). Expression from each shRNA        cassette is driven by a U6 promoter. Cell cultures were        maintained for up to 10 additional days in X-VIVO15 medium        supplemented with 5% FBS and 30 ng/m1 of IL-2. On d4, 7, and/or        10 post-nucleofection, the cultures were sampled and analyzed        for surface expression of CD3 (anti-CD3-BV711, BioLegend), CD34        (anti-CD34-PE, or APC, LifeTechnologies), B2M (anti-B2M-APC, or        PE, BioLegend), and/or HLA-A, B, and C (clone W6/32, BV605).        Flow cytometry data were acquired on a Beckman-Coulter        CytoFLEX-LX.

2. Results

B2M surface levels were measured in samples nucleofected with a controlCAR construct (7002) or with CAR constructs expressing one (7056 or7059) or two (7060) copies of B2M-specific shRNA in either head-to-tail(7056) or tail-to-tail (7059, 7060) configurations. A restriction digestsite that was present in previous constructs between the U6 promoter andthe shRNA sequence was been removed from these shRNA472 vectors. It washypothesized that the palindromic restriction digest site interferedwith the efficacy of the constructs and the ability of the shRNA toknock down B2M.

When comparing the CD3−/CD34+ populations in 7002 (control) and 7056(single shRNA) expressing cells, 7056 expressing cells were observed todisplay lower levels of surface B2M (77% knockdown) (FIG. 13A). Cellsnucleofected with 7059 (single copy, tail-to-tail) displayed a 90.1%knockdown of B2M relative to 7002 control cells (FIG. 13B), while 7060nucleofection (two copies, tail-to-tail) resulted in a 92% knockdownrelative to 7002 controls (FIG. 13C).

3. Conclusions

A pre-screened B2M-targeting shRNA can knock down B2M expression levelson the surface of cells into which the construct has been delivered (viatargeted insertion into the T cell receptor alpha constant regionlocus). Removing the cloning site between the U6 promoter and thehairpin sequence improves the efficiency with which B2M is knocked down.7008 (tail-to-tail, one shRNA472 cassette—FIG. 12A) supports minimal B2Mknockdown while 7059 (one cassette, tail-to-tail—FIG. 13B) supportsgreater than 90% knockdown. As was observed using a CD52-specific shRNA,superior knockdown was observed when the CAR promoter and the shRNApromoter were oriented in different directions (tail-to-tailconfiguration). Adding a second shRNA sequence did not provide anynoticeable benefit (92% versus 90.1% knockdown).

EXAMPLE 5 Production and Characterization of CAR T Cells Utilizing shRNAto Reduce Cell Surface Expression of B2M 1. Materials and Methods

In this study, an apheresis sample was drawn from a healthy, informed,and compensated donor, and the T cells were enriched using the CD3positive selection kit II in accord with the manufacturer's instructions(Stem Cell Technologies). T cells were activated using ImmunoCult T cellstimulator (anti-CD2/CD3/CD28—Stem Cell Technologies) in X-VIVO 15medium (Lonza) supplemented with 5% fetal bovine serum and 10 ng/ml IL-2(Gibco). After 3 days of stimulation, cells were collected and samplesof 1e6 cells were electroporated with lug of RNA encoding the TRC1-2L.1592 meganuclease, which recognizes and cleaves the TRC 1-2recognition sequence in the T cell receptor alpha constant locus, andwere transduced with AAV packaged with construct 7056 at an MOI of 25000viral genomes/cell.

Cell cultures were maintained for up to 10 additional days in X-VIVO15medium supplemented with 5% FBS and 30 ng/ml of IL-2. On day 4, 7,and/or 10 post-nucleofection, the cultures were sampled and analyzed forsurface expression of CD3 (anti-CD3-PE, BioLegend), (anti-FMC63 anti-CARclone VM16 conjugated to AlexaFluor488), B2M (anti-B2M-APC, or PE,BioLegend), and HLA-A, B, and C (clone W6/32, BV605). Flow cytometrydata were acquired on a Beckman-Coulter CytoFLEX-LX.

2. Results

B2M and HLA-ABC levels were measured in samples expressing construct7056 and control populations. FIG. 14A shows the B2M surface levels inCD3-/CAR+ cells compared to TRAC-edited cells expressing no shRNA from acontrol culture. FIG. 14B shows B2M levels on CD3-/CAR+ versus CD3+/CAR−populations in the same culture. FIG. 14C and 14D make the samerespective comparisons in displays of HLA-ABC surface levels. TheCD3-/CAR+ fraction of cells transduced with AAV-7056 displayed levels ofB2M and HLA-ABC that are reduced by greater than 90% compared to controlpopulations.

3. Conclusions

A pre-screened B2M-targeting shRNA can knock down B2M expression levelson the surface of cells into which the construct has been delivered (viatargeted insertion into the T cell receptor alpha constant regionlocus). This effect is specific to CAR+ populations (i.e., cells inwhich targeted integration into the TRAC locus has occurred). Thisexperiment demonstrates that B2M can be efficiently knocked down using asingle copy of shRNA472 co-delivered to the TRAC locus with the CAR geneon the same AAV template.

EXAMPLE 6 Characterization of Candidate shRNAs Against CD52 in PrimaryHuman T Cells 1. Materials and Methods

Five Mission-shRNA lentiviral transfer plasmids encoding different CD52targeting sequences were purchased from Sigma-Aldrich. Second-generationlentiviral vectors were produced in-house using Lenti-X 293T cells(ClonTech) and a triple transfection method (Lipofectamine2000—Thermo-Fisher). T cells were prepared for lentiviral transductionby stimulating for 3 days with ImmunoCult anti-CD2/CD3/CD28 as inExample 1. Transduction was carried out in the presence of 5 uMpolybrene (Sigma-Aldritch) and transduced cells were expanded for 5 daysin IL-2 supplemented medium before a flow cytometric analysis of CD52surface levels. Cells were not selected with puromycin because aheterogeneous population was desired for downstream attempts at magneticdepletion of CD52Hi cells. Cells transduced with a lentivirus encodingshRNA 568 were labeled with biotinylated anti-CD52 (Miltenyi Biotec),and magnetic separation was performed using a Biotin Positive SelectionKit (StemCell Technologies). A post-separation analysis of surface CD52was performed.

2. Results

Of the 5 shRNA sequences screened, 3 (shRNA568, shRNA572, and shRNA876)interfered with CD52 expression. CD52 surface expression profiles aredisplayed in FIG. 8. Levels of CD52 displayed on the surface of T cellsare shown in FIG. 9 for mock transduced T cells (9A), T cells transducedwith an shRNA-568 lentivirus (9B), and LV-shRNA568 transduced cells thathave undergone a CD52 magnetic depletion (9C).

3. Conclusions

CD52 antigen density on the surface of cells can be reduced using shRNAdelivered by a viral vector. Sequence 568 exhibited the highest degreeof CD52 knockdown. Knockdown of CD52 using this shRNA sequence wassufficient to allow for magnetic depletion of non-transduced CD52 Hicells.

EXAMPLE 7

CD52 Knockdown Profiles Using CAR/CD52 Constructs with DifferentOrientations

1. Materials and Methods

T cells were stimulated for 3 days using ImmunoCult anti-CD2/CD3/CD28 asdescribed in EXAMPLE 1. After 3 days, TRC 1-2x.87EE mRNA, STING siRNA,and linearized AAV transfer vector encoding different CAR constructs(FIG. 10) were delivered to the T cells using the 4-D Nucleofector(Lonza). Cultures of nucleofected T cells were carried for 10 days inmedium supplemented with IL-2 prior to flow cytometric analyses of CD3,CAR (CD34 epitope-tagged), and CD52.

2. Results

To demonstrate un-manipulated levels of CD52 surface display onTRAC-edited CAR T cells, a TRC 1-2x.87EE nuclease and a CD34-tagged CARconstruct encoding no shRNA sequence were delivered. CD52 levels on TCRKO cells, TCR KO CAR+ cells and nonedited cells are overlaid in thehistogram in FIG. 11A. Three CAR constructs encoding a U6promoter-controlled CD52 shRNA were evaluated for ability to knock downCD52 when integrated into the TRAC locus. When the CAR gene and theshRNA cassette are both in forward orientation, CD52 antigen density isreduced by approximately 50% (FIG. 11C; construct 7004). Reversing thetranscriptional orientation of the CAR gene alone (i.e., a tail-to-tailconfiguration) reduces the amount of CD52 displayed on the surface byapproximately 95% (FIG. 11B; construct 7013), while reversing theorientation of both the CAR gene and the U6-shRNA element reduces theCD52 signal by approximately 90% (FIG. 11D; construct 7014).

3. Conclusions

The CD52 specific shRNA sequence 568 can interfere with CD52 expressionwhen only one copy is delivered by targeted insertion into the TRAClocus. Altering the transcriptional orientation of either the CAR geneonly (i.e., tail-to-tail configuration) or both the CAR and shRNA genescan influence the efficiency of target gene knockdown. Reversing onlythe CAR gene's orientation resulted in the most efficient knockdown.

4. Further Studies

A number of constructs were prepared comprising an anti-CD19 CAR codingsequence and an shRNA against CD52. These are illustrated in FIG.10A-10H and are provided in SEQ ID NOs: 10-17. As described above, CARconstructs 7004 (SEQ ID NO: 12), 7013 (SEQ ID NO: 14), and 7014 (SEQ IDNO: 16) were previously evaluated for their ability to reduce CD52expression while expressing a CAR. The 5′ and 3′ homology arms flankingthe CAR coding sequence and the shRNA sequence have homology to regionsupstream and downstream of the TRC 1-2 recognition sequence in the TRAClocus.

In additional studies, CAR T cells will be prepared using primary donorhuman T cells transduced with recombinant AAV vectors comprising one ofthe CAR/shRNA constructs above, with simultaneous nucleofection of mRNAencoding the TRC 1-2x.87EE to induce a double-strand break at the TRC1-2 recognition sequence and promote targeted insertion of the constructinto the genome of the T cells. CD52 expression will be determined asdescribed above to determine which orientation of the first and secondexpression cassettes will result in the highest and/or the mostconsistent CAR expression, along with the most consistent level of CD52knockdown on the cell surface.

CAR T cells produced with certain constructs will be evaluated in boththe allogenicity and NK cell killing assays previously described above.Further, CAR T cells produced using the disclosed constructs will beevaluated in various stress tests, in which the CAR T cells arerepeatedly exposed to antigen in order to determine changes in cellproliferation/expansion and cytotoxic potential. CAR T cells producedusing the disclosed constructs will also be utilized with in vivo tumormodels to determine their ability to clear tumor cells in an animal andto evaluate their ability to persist in vivo. It is expected, based onthe Examples described herein, that enriched populations of CAR T cellscan be produced for in vivo use by an advantageous negative-selectionfor CAR T cells having reduced cell surface expression of CD52.

1. A nucleic acid molecule comprising: (a) a first expression cassettecomprising a nucleic acid sequence encoding an engineered antigenreceptor; (b) a second expression cassette comprising a nucleic acidsequence encoding an inhibitory nucleic acid molecule; (c) a 5′ homologyarm; and (d) a 3′ homology arm; wherein said 5′ homology arm and said 3′homology arm have homology to chromosomal regions flanking a nucleaserecognition sequence in a gene of interest.
 2. The nucleic acid moleculeof claim 1, wherein said inhibitory nucleic acid molecule is an RNAinterference molecule.
 3. The nucleic acid molecule of claim 1, whereinsaid nuclease recognition sequence comprises SEQ ID NO:
 1. 4. Thenucleic acid molecule of claim 1, wherein said inhibitory nucleic acidmolecule is an shRNA inhibitory against beta-2 microglobulin, whereinsaid shRNA has a sequence comprising any one of SEQ ID NOs: 2-4.
 5. Thenucleic acid molecule of claim 4, wherein said shRNA has a sequencecomprising SEQ ID NO:
 2. 6. The nucleic acid molecule of claim 4,wherein said first expression cassette and said second expressioncassette are in a 3′ to 5′ orientation relative to said 5′ and 3′homology arms, and wherein said first expression cassette is 5′ upstreamof said second expression cassette and wherein said first expressioncassette comprises: a nucleic acid sequence encoding a chimeric antigenreceptor or an exogenous T cell receptor; (ii) a JeT promoter whichdrives expression of said chimeric antigen receptor or said exogenous Tcell receptor; and (iii) a polyA sequence; and wherein said secondexpression cassette comprises: (iv) a nucleic acid sequence encodingsaid shRNA; (v) a U6 promoter which drives expression of said shRNA; and(vi) a central polypurine tract and central terminator sequence(cPPT/CTS) sequence.
 7. A genetically-modified eukaryotic cellcomprising said nucleic acid molecule of claim 1, wherein saidengineered antigen receptor and said inhibitory nucleic acid moleculeare expressed in said genetically-modified eukaryotic cell.
 8. Thegenetically-modified eukaryotic cell of any one of claims 7, whereinsaid inhibitory nucleic acid molecule is inhibitory against human beta-2microglobulin.
 9. The genetically-modified eukaryotic cell of claim 8,wherein said genetically-modified eukaryotic cell is agenetically-modified human T cell, and wherein said engineered antigenreceptor is a chimeric antigen receptor or an exogenous T cell receptor.10. The genetically-modified eukaryotic cell of claim 7, wherein saidinhibitory nucleic acid molecule is inhibitory against human CD52.
 11. Agenetically-modified eukaryotic cell comprising in its genome a nucleicacid sequence encoding an engineered antigen receptor which is expressedby said genetically-modified eukaryotic cell, wherein cell surfaceexpression of beta-2 microglobulin on said genetically-modifiedeukaryotic cell is reduced by 10% to 95% compared to cell surface beta-2microglobulin expression on a control cell.
 12. A genetically-modifiedeukaryotic cell comprising in its genome a nucleic acid sequenceencoding an engineered antigen receptor which is expressed by saidgenetically-modified eukaryotic cell, wherein cell surface expression ofMHC class I molecules on said genetically-modified eukaryotic cell isreduced by 10% to 95% compared to cell surface expression of MHC class Imolecules on a control cell.
 13. The genetically-modified eukaryoticcell of claim 11, wherein said genetically-modified eukaryotic cell is agenetically-modified human T cell.
 14. The genetically-modifiedeukaryotic cell of claim 13, wherein said genetically-modified human Tcell expresses a chimeric antigen receptor or an exogenous T cellreceptor.
 15. A method for producing a genetically-modified eukaryoticcell, said method comprising introducing into a cell said nucleic acidmolecule of claim 1 and: (a) a nucleic acid encoding an engineerednuclease having specificity for said nuclease recognition sequence,wherein said engineered nuclease is expressed in said cell; or (b) anengineered nuclease protein having specificity for said nucleaserecognition sequence; wherein said engineered nuclease recognizes andcleaves said nuclease recognition sequence in the genome of said cell togenerate a cleavage site, and wherein said nucleic acid molecule isinserted into the genome of said cell at said cleavage site.
 16. Themethod of claim 15, wherein said inhibitory nucleic acid molecule isinhibitory against human beta-2 microglobulin.
 17. A method of usingimmunotherapy to treat a disease in a subject in need thereof, saidmethod comprising administering to said subject a therapeuticallyeffective amount of said genetically-modified eukaryotic cell of claim9; wherein said genetically-modified eukaryotic cell is agenetically-modified human T cell expressing a chimeric antigen receptoror an exogenous T cell receptor; and wherein cell surface expression ofbeta-2 microglobulin on said genetically-modified human T cell isreduced by 10% to 95%, by 50% to 95%, by 75% to 95%, or by 90% to 95%compared to cell surface beta-2 microglobulin expression on a controlcell.
 18. A method of using immunotherapy to treat a disease in asubject in need thereof, said method comprising administering to saidsubject a therapeutically effective amount of said genetically-modifiedeukaryotic cell of claim 14; wherein said genetically-modifiedeukaryotic cell is a genetically-modified human T cell expressing achimeric antigen receptor or an exogenous T cell receptor; and whereincell surface expression of WIC class I molecules on saidgenetically-modified human T cell is reduced by 10% to 95%, by 50% to95%, by 75% to 95%, or by 90% to 95% compared to expression of WIC classI molecules on a control cell.
 19. A method of using immunotherapy totreat a disease in a subject in need thereof, said method comprisingadministering to said subject a therapeutically effective amount of saidgenetically-modified eukaryotic cell of claim 10; wherein saidgenetically-modified eukaryotic cell is a genetically-modified human Tcell expressing a chimeric antigen receptor and an inhibitory nucleicacid against CD52; and wherein cell surface expression of CD52 on saidgenetically-modified human T cell is reduced by 10% to 95%, by 50% to95%, by 75% to 95%, or by 90% to 95% compared to cell surface CD52expression on a control cell.
 20. A method for preparing an enrichedpopulation of genetically-modified eukaryotic cells comprising anengineered antigen receptor, said method comprising preparing apopulation of cells comprising said genetically-modified eukaryotic cellof claim 70, and cells expressing a wild-type level of cell surfaceCD52, wherein cell surface expression of CD52 on saidgenetically-modified cell is reduced by 10% to 95%, by 50% to 95%, by75% to 95%, or by 90% to 95% compared to cell surface CD52 expression ona control cell, said method comprising: (a) contacting said populationof cells with beads conjugated to an anti-CD52-binding molecule, whereincells expressing a wild-type level of cell surface CD52 are bound tosaid beads and said genetically-modified eukaryotic cell is not bound tosaid beads; and (b) removing said beads from said population of cells toproduce said enriched population of cells; wherein said enrichedpopulation of cells is enriched for said genetically-modified eukaryoticcell.